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. 2010 Sep;24(9):3438–3450. doi: 10.1096/fj.10-154765

Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule

Ming Chang Hu *,†,‡, Mingjun Shi *, Jianning Zhang , Johanne Pastor §, Teruyo Nakatani , Beate Lanske , M Shawkat Razzaque , Kevin P Rosenblatt §, Michel G Baum †,‡, Makoto Kuro-o *,§, Orson W Moe *,†,‖,1
PMCID: PMC2923354  PMID: 20466874

Abstract

Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho’s known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant β-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by β-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.—Hu, M. C., Shi, M., Zhang, J., Pastor, J., Nakatani, T., Lanske, B., Shawkat Razzaque, M., Rosenblatt, K. P., Baum, M. G., Kuro-o, M., Moe, O. W. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

Keywords: renal brush border membrane, FGF23, NaPi-2a, β-glucuronidase, proteolytic degradation


Klotho was originally identified as an antiaging gene because Klotho hypomorphs and Klotho knockout (Kl−/) mice exhibit multiorgan premature aging (1). Reconstitution of Klotho protein into the Kl−/− background mice through genetic manipulation (1, 2) or adenoviral transfer (3) rescues most of phenotype in Kl−/− mice. The other striking feature in Klotho-deficient mice is mineral disturbances, including hyperphosphatemia, hypercalcemia, vascular calcification, and elevated 1,25(OH)2 vitamin D3 and fibroblast growth factor (FGF23) (1, 4, 5). The mechanisms of the deranged mineral metabolism are not understood. The full spectrum of human mutations of Klotho is not yet known, but the missense mutation causes multiple mineral disturbances, including severe hyperphosphatemia (6) and a translocation resulting in possible overexpression of Klotho causes hypophosphatemia (7).

Recent studies ushered in novel molecular mechanisms, whereby Klotho controls renal calcium homeostasis. Chang et al. (8)proposed that Klotho activates the transient receptor potential v-5 (TRPV5) as a β-glucuronidase entrapping the channel in the plasma membrane to enhance calcium reabsorption in the distal nephron. Cha et al. (9) showed that TRPV5 retention is due to removal of sialic acid, enabling the TRPV5 glycan to bind to galectin-1 to stabilize its membrane residence. A model was submitted by Imura et al. (10) of intracellular binding of the Na-K-ATPase α1 subunit by Klotho, which increases plasma membrane pump activity, which activates basolateral calcium exit via the sodium calcium exchanger (NCX)-1. While models for modulation of calcium transport in the distal nephron continue to unravel, the mechanisms of how Klotho regulates phosphate have not been addressed.

Although disturbances of plasma phosphate (Pi) concentrations can be transiently caused by extracellular-intracellular shifts, sustained hyperphosphatemia invariably reflects disturbance in external balance, which is controlled largely by renal excretion. The kidney handles Pi via sequential glomerular filtration and reabsorption, almost exclusively by the proximal tubule, mediated principally by apical membrane sodium-coupled Pi transporters NaPi-2a, NaPi-2c (11, 12), and one isoform of NaPi-3 called Pit-2 (13,14,15), which are targets of regulation by multiple phosphaturic hormones (16). Klotho-deficient mice displayed increased activity and levels of the NaPi-2a and NaPi-2c transporters compared with wild-type (WT) mice (4). This suggests that the hyperphosphatemia, at least in part, stems from renal origin, perhaps through regulation of NaPi cotransporters in kidney.

However, complex multifaceted systemic changes in mineral metabolism in animals with chronic alterations in Klotho preclude such conclusions. Klotho has been suggested to function as an enzyme (8, 9, 17) on apical proteins facing the urinary lumen, although how and whether Klotho gains access to the urinary lumen is unknown. Since Klotho also functions as a coreceptor for FGF23 (18,19,20), it is unclear whether Klotho simply enhances the FGF23 effect or functions as a direct phosphaturic substance, and if so, by what mechanisms Klotho exerts this action. This work seeks to address the above questions using both in vivo and in vitro models. In this study, we showed that Klotho could function as a phosphaturic substance independently from FGF23 via its enzymatic action on renal NaPi-2a involving glucuronidase activity, resulting in inhibition of transporter activity, proteolytic degradation, and eventually reduced surface expression of NaPi-2a, possibly via internalization.

MATERIALS AND METHODS

Animals

Transgenic mice overexpressing of Klotho (Tg-Kl; line EFmKL46) and Klotho hypomorph mice (Kl−/−) were cared for at the Animal Research Center at the University of Texas Southwestern Medical Center. The appropriate strains of wild-type littermates were used as controls for Tg-Kl or Kl−/−, respectively. The age of mice ranged from 6 to 8 wk. Three days prior to termination, mice were transferred to individual metabolic cages for 2 d of adaptation. Twenty-four-hour urine samples were collected, blood was drawn through cardiac puncture for plasma chemistries, and kidneys were harvested. Normal Sprague-Dawley (SD) rats (220–250 g body weight) were purchased from Harlan (Indianapolis, IN, USA). Under Inactin anesthesia, 64 pmol of full extracellular domain of recombinant mouse Klotho protein (rMKl) (2) was intravenously administrated once via the jugular vein. Urine of rats was collected through bladder catheterization for the duration indicated. In some studies, rat kidneys were perfused with fixative via aortic puncture, and then kidneys were removed for immunohistochemistry. FGF23-null mice (16–20 g) were maintained in Animal Research Center at the Harvard School of Dental Medicine (21). Anesthetized mice were intraperitoneally injected with 6.0 pmol of rMKl once. All animal work was approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center or Harvard Medical School.

Plasma and urine biochemistry

Plasma and urinary chemistries were analyzed by the Mineral Metabolism Core laboratory in the Pak Center at Southwestern using Beckman CX9ALX; Beckman Coulter (Fullerton, CA, USA). Urinary phosphorus was assayed by a Roche COBAS MIRA autoanalyzer (Roche Phosphomolybdate UV kit; Roche Diagnostics, Mannheim, Germany); calcium by atomic absorption using a Varian SpectrAA 220 (Varian, Palo Alto, CA, USA) and SIPS autosampler; sodium/potassium by flame emission using an Instrumentation Laboratory Flame Photometer 943; and creatinine using a Roche kinetic alkaline picrate kit on a Roche COBAS MIRA autoanalyzer. Blood and urinary glucose was determined using the Vitros 250 Chemistry System. Urinary amino acid was detected with thin-layer chromatography (cellulose plate) (Sigma-Aldrich, St. Louis, MO, USA). Standard amino acid solution (Sigma-Aldrich) was used for positive control. Ten microliters of standard and urine samples were loaded on cellulose plates for chromatography, followed by 0.2% ninhydrin staining (Sigma-Aldrich, St. Louis, MO, USA).

RT-PCR

Total RNA was extracted using the RNAeasy kit (Qiagen, Valencia, CA, USA) from an SV40-immortalized mouse DCT cell line (22), and different nephron segments, including glomeruli, proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs), and cortical collecting ducts (CCDs) obtained by microdissection (23). Complementary DNA (cDNA) was generated with oligo-dT primers using the SuperScript III First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Membrane and secreted forms of mouse Klotho, and mouse β-actin were detected using primers previously described (24). Primers 5′-CCAGCACTGCCACCATAACCATTT-3′ and 5′-AATGCGCCTCTCCTC-CTCTTCTTT-3′; and 5′-AATCTCGAGATCGGATCCTG-3′ and 5′-CTCTGTTCCA-AGGACTGCAT-3′ were used for detection of NCX1 and NHE3 mRNA, respectively: PCR products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide.

Opossum kidney (OK) cells and transient transfection

OK cells were used as an in vitro model as they exhibit biological features of proximal tubules, including expression of native NaPi-2a (25, 26). OK cells were cultured and maintained as described previously (27). For transient transfections, OK cells were grown to ∼70% confluence, and 1.0 μg of cDNA was introduced per 35-mm plate with Lipofectamine Plus (Invitrogen) following product instructions. Transfection efficiency was monitored by empty eGFP-C1 vector (Clontech, BD, Palo Alto, CA, USA) and evaluated by fluorescent microscopy (typically 70–80% efficiency). Experiments were performed at 48 h post-transfection.

To evaluate NaPi-2a trafficking, OK cells were seeded on glass coverslips as described previously (27) and transfected with WT or mutant OK NaPi-2a/eGFP. Two days post-transfection, OK cells were incubated with Klotho or vehicle for the indicated duration and dose, washed with PBS, and fixed with 4% paraformaldehyde (10 min), followed by another rinse with PBS. Cells were permeabilized with 0.1% Triton X-100 (10 min at 4°C), rinsed, and then incubated with rhodamine-phalloidin (1:50) (Molecular Probes, Eugene, OR, USA) to stain β-actin filaments. Confocal fluorescent images were visualized through a Zeiss ×100 objective lens using a Zeiss LSM-510 laser-scanning confocal microscope(Carl Zeiss, Oberkochen, Germany). To obtain the live image of NaPi-2a/eGFP, OK cells transiently transfected with NaPi-2a/eGFP were incubated in 37°C chamber filled with 5% CO2. The NaPi-2a/eGFP traffic under the effect of Klotho protein (0.4 nM) or vehicle was visualized, consistently scanned for 4 h at 30-minute intervals, and analyzed with Zeiss LSM image software.

Recombinant mouse Klotho protein

Soluble Klotho protein containing the entire extracellular domain of mouse Klotho (amino acid number 31–982) with C-terminal V5 and 6xHis tags were purified from conditional medium by affinity column chromatography using anti-V5 antibody (Sigma-Aldrich), as previous described (2). “Mutant Klotho” has 6 mutations of putative active sites in Asp-240, Asn-242, Glu-416, Asn-417, Asn-690, and Glu-691 to Ala (2, 9). WT and mutant Kotho in conditioned medium (CM) were prepared from 293 stable cell lines transfected with WT or mutated Klotho, as described previously (2, 9).

Mutagenesis of asparagines on OK NaPi-2a

The full-length coding region of OK NaPi-2a was cloned by PCR into the plasmid peGFP/C1 (Clontech), yielding WT-OK-NaPi-2a/eGFP. The mutations were sequentially introduced at N300 and then N332 to generate doubly mutated OK-NaPi-2a/eGFP/N300Q/N332Q using QuickChange site-directed mutagenesis Kit (Stratagene, La Jolla, CA, USA) following product instructions. The primers used for mutagenesis of N300Q and N332Q of OK-NaPi-2a were 5′-GAGATGAGACCCTTCGAcAgCACAGCCTYCATCCGGATT-3′ and 5′-TCAGCTGGACCCTGGGGcAgACCACTGGGGAGAAATGT-3′, respectively. We used primers 5′-CGCGGATCCATGTACCCATACGATGTTCCAGATTACGCTATGATGCCTTACAGAGAGAGA-3′ and 5′-CCGGAATTCTTAGAGCCTAGTGGCGTTGTG-3′ to construct WT HA/OK-NaPi-2a in pCDNA3.1 and then used the same primers of mutagenesis to construct DM HA/OK-NaPi-2a. The WT and mutated sequences were verified by direct sequencing using Applied Biosystems Inc. (Foster City, CA, USA) Big Dye Terminator 3.1 chemistry.

In vivo micropuncture study and protein microarray

Collection of luminal fluid from PCTs and DCTs was carried out by in vivo free-flow micropuncture on SD rats as described previously (28, 29). Briefly, under Inactin anesthesia, left kidney was exposed and placed on the micropuncture setup, and the left ureter was catheterized for urine collection. PCTs were identified by their characteristic configuration after lissamine green dye injection and punctured with sharpened glass capillaries with a diameter of 7–8 μm. Tubular fluid from PCTs (∼60–120 nl) and DCTs (40–80 nl) was collected over 10–12 min. After the volume was measured in a calibrated constant-bore glass capillary, the fluid was loaded on nitrocellulose-coated FAST slides (Schleicher & Schuell BioScience, Inc. Keene, NH, USA) for protein microarray, as described previously (30). Slides were probed with anti-Kl1 (KM2076) antibody followed by amplification using a tyramide-based avidin/biotin system (catalyzed signal amplification system; Dako, Glostrup, Denmark). A pegylated, streptavidin-conjugated Quantum Dot 655-Sav (Quantum Dot Corp., Carlsbad, CA, USA) was used as a final fluorophore. Slides were visualized using Alpha Innotech’s 9900 Fluorimeter with 655-nm narrow bandwidth emission filter (Omega Optical, Brattleboro, VT, USA).

In vitro microperfusion study

Single PCTs were microdissected on ice from kidney cortex slices of Tg-Kl mice and their WT littermates and were set up for in vitro microperfusion (31, 32). Isolated segments of superficial PCTs were perfused and PCT phosphate flux (JPhos) was calculated as described previously (31, 32). For immunostaining of NaPi-2a protein in single PCTs, some PCTs were incubated with 0.4 nM rMKl protein in perfusion solution or at bath chamber for 30 min. After perfusion, the tubules were fixed and transferred to the gelatin, as described previously (33). The gelatin droplets containing the tubules were washed in PBS, cut into cubes, and frozen at −80°C until use.

Brush border membrane vesicle (BBMV) preparation

Kidney cortexes were immediately dissected in ice-cold isolation buffer after being removed from animals and then homogenized using a Potter-Elvehjem homogenizer at 4°C). BBMVs were prepared using three consecutive magnesium precipitations (15 mM) (32, 34). To verify brush border membrane (BBM) purify, the activity of Na-H exchanger (35), Na-dependent citrate cotransporter, and the activity of BBM enzyme, including maltase, alkaline phosphatase, and leucine aminopeptidase in samples of cortical homogenates and BBM fractions were regularly monitored (34). BBMVs would be used for Na-dependent Pi uptake assay and for immunoblot.

Na-dependent Pi uptake by BBMVs or OK cells

Uptake of Na-dependent 32P by freshly isolated BBMVs (200 μg protein/filter at 10 μg/μl) was measured by the Millipore filtration technique as described previously (36). Final concentration of phosphate was 0.1 mM. Uptake was terminated after 15 s by rapid addition of ice-cold stopping solution (100 mM NaCl, 10 mM mannitol, 16 mM HEPES, and 10 mM Tris-HCl, pH 7.5) followed by filtration. For each data point, the uptake was measured in triplicate at each time period.

OK cells were grown in 24-well plates and incubated with Klotho and/or FGF23 for the indicated time/dose followed by Na+-dependent uptake of 32P-phosphate by OK cells (37). Briefly, cells were rinsed with Na+-free solution followed by incubation with uptake solutions containing 0.1 mM phosphate (1 μCi/ml, Amersham Life Sciences, Little Chalfont, UK) with or without 140 mM Na+ for 5 min. The reaction was stopped by aspiration of uptake solution; cells were washed with ice-cold stop solution (140 mM NaCl, 1 mM MgCl2, 10 mM HEPES pH 7.4). Each transport reaction was measured in triplicate.

Measurement of surface NaPi-2a in OK cells

Because antibody against native OK NaPi-2a protein was not available to us, we used OK cells transiently transfected with NaPi-2a tagged with HA and used monoclonal HA antibody (Sigma) to detect HA/NaPi-2a protein. Biotinylation for surface protein was conducted following our previous protocol (27), but with key modifications as stated below. NHS-PEO-maleimide (Pierce, Rockford, IL, USA) was used. OK cells were treated with 5 mM tris-(2-cartoxy-ethyl)-phosphine hydrochloride (TCEP; Sigma) (4°C for 10 min) to reduce SH group, then biotinylated with 1 mM biotin maleimide (4°C for 60 min) to cross-link cysteine residues, followed by quenching free maleimide with 50 mM cysteine (4°C for 20 min).

Immunoblot

Cell lysate, kidney cortex homogenate, or BBMVs for immunoblot were made as described previously (27, 32). Thirty micrograms of protein was solubilized in Laemmli sample buffer, electrically fractionated on SDS-PAGE, transferred to PVDF membrane, and subjected to immunoblot using specific antibodies, including polyclonal rabbit antibody for NaPi-2a (1:3000 dilution), monoclonal rat antibody for human Kl1 (KM2076) (1:1000), polyclonal rabbit antibody for human Klotho (KMDC) (1:3000), monoclonal mouse antibody for β-actin (1:5000), and monoclonal mouse antibody for HA(1:500). Primary antibodies were incubated overnight at 4°C. After washing, membranes were incubated with the secondary antibodies conjugated with horseradish peroxidase (Amersham Life Sciences). Specific signal was visualized by ECL kit (Amersham Life Sciences).

Kidney immunohistochemistry

In some experiments, for clear visualization of signal in proximal tubules, kidneys were fixed in situ with perfusion of 2.5% paraformaldehyde via the aorta before kidneys were removed, while in other experiments, kidneys were harvested and directly frozen in optimal cutting temperature (OCT) compound (Tissue TeK; Sakura Finetek, Torrance, CA, USA) using liquid N2. Kidney cryosections (4 μm) were made and subjected to immunofluorescence staining as described previously (33) or kept at −20°C until use. Polyclonal rabbit antibody for NaPi-2a (1:300 dilution) or monoclonal rat antibody for Kl1 (KM2076) (1:200) followed by secondary antibodies conjugated to fluorescein isothiocyanate or rhodamine (Molecular Probes, Eugene, OR, USA) were used for double staining. Sections were visualized with a Zeiss LSM510 microscope.

Immunoelectron microscopy

The kidneys were fixed with 2.5% paraformaldehyde via aortic perfusion, removed, and postfixed in 4% paraformaldehyde at 4°C for 4 h. Immunogold labeling of ultrathin frozen tissue sections was performed according to Tokuyasu’s method (38). Kidney cortex was infiltrated with 2.3 M sucrose overnight, frozen in liquid nitrogen, and 70- to 80-nm-thick sections were cut on a ultracryomicrotome (Ultramicrotome Reichert Ultracut E; Leica Microsystems, Wetzlar, Germany) and mounted on Formvar-coated nickel grids. The sections were incubated with Kl1 rabbit antibody (KMDC1) (1:20) for 60 min after rehydration and blocking; and followed by incubation with gold-conjugated protein A (10-nm gold particles, Sigma-Aldrich) for 60 min. After staining with uranyl acetate, sections were visualized with Jeol 1200 EX transmission electron microscope (Jeol Ltd., Akishima, Japan).

Statistical analyses

Data are expressed as means ± se. Statistical analysis was performed using an unpaired Student’s t test, or ANOVA followed by Student-Newman-Keuls test whenever appropriate. A value of P ≤ 0.05 was considered as statistically significant. Unless otherwise stated, representative figures reflect the results in a minimum of 3 independent experiments.

RESULTS

Klotho mRNA and protein in renal proximal tubules

To understand how Klotho affects Pi transport by the proximal tubule in the kidney, it is important to establish how Klotho gains access to the proximal tubule other than the presumed hematogenous route (unpublished results). Glomerular filtration is highly unlikely because of its size (130 kDa). Klotho is expressed in tubules in the renal cortex in WT mice, whereas transgenic mice overexpressing Klotho (Tg-Kl) have increased expression, as well as ectopic expression, of Klotho in a variety of tissues (1, 2) and in a variety of nephron structures (Supplemental Fig. 1A, B). In contrast, Kl−/− mice had barely detectable Klotho compared to WT mice (Supplemental Fig. 1A, B). There is strong expression of Klotho protein in the DCT, colocalizing with the sodium-calcium exchanger NCX1 (Fig. 1A, right panel), concordant with previous studies (39). We also detected definite, albeit weaker, expression in the PCT, colocalizing with NaPi-2a protein (Fig. 1A left panel). To further confirm its presence in the PCT, we immunostained single microdissected mouse PCTs and found Klotho protein in the cytoplasm and apical and basolateral membranes (Supplemental Fig. 1C). Immunogold electron microscopy confirmed the immunohistochemical finding (Fig. 1BE). Theoretically, Klotho protein can be delivered to (from the capillary or distal tubule) and taken up by the PCT on its basolateral side, synthesized in situ in the PCT, or both. RT-PCR in microdissected nephron segments showed Klotho mRNA in the DCT (coamplified with NCX1), and in the PCT (coamplified with NHE3) (Fig. 1F). This definitely supports synthesis of Klotho in the proximal tubule, although we cannot rule out some proximal tubule uptake of Klotho. Full-length Klotho protein (130 kDa) was detected in fresh urine from mice (Fig. 1G), rats, and humans (Supplemental Fig. 1D). To examine whether Klotho protein is present in the luminal proto-urine of the PCT, we collected tubular fluid from the PCT by in vivo free-flow micropuncture and detected Klotho antigen in proximal proto-urine from WT but not from Kl−/− mice (Fig. 1H). We unequivocally demonstrated that in the PCT, Klotho mRNA is transcribed and Klotho protein is present in the proximal tubular cell, apical and basolateral membrane, and proximal urinary lumen, placing it at an ideal locale for regulating Pi transport.

Figure 1.

Figure 1.

Klotho protein and mRNA expression in the kidney, proximal tubular fluid, and urine of mice. A) Representative coexpression of Klotho (green) with NaPi-2a (red) in proximal tubule and with NCX1 (red) in distal tubule (examined in a total of 3 mouse kidneys). Kidney sections of mice were stained with rat anti-Klotho antibody (KM2076) and with rabbit anti-NaPi-2a serum. B–E) Immunoelectron microscopy of Klotho (arrows) in the distal tubule (B, C) and proximal tubule (B, D, E). Klotho protein was detected in the basolateral membrane (B) and the apical brush border (E), as well as the cytoplasm (C) of the proximal tubule. In the distal tubule, Klotho protein is highly expressed in cytoplasm (B, C), the basolateral membrane (B), and the tubular lumen (C). Klotho protein is also present in the capillary lumen adjacent to distal tubules (B). Endo, endothelium of capillary; MV, microvilli; TBM, tubular basement membrane. Similar results were obtained from 3 animals (kidneys). F) RT–PCR of Klotho transcript in microdissected glomeruli and proximal and distal tubules. Na/H exchanger 3 (NHE3, 237 bp) and Na/Ca exchanger 1 (NCX1, 553 bp) served as markers for proximal and distal tubules, respectively, and β-actin (453 bp) served as internal control. Similar results were obtained from 3 animals (samples). G) Klotho immunoreactivity in fresh urine of mice. Lanes 1 and 2 were loaded with 20 and 10 fmol of recombinant mouse Klotho (rMkl), respectively; Lanes 3 and 4 were loaded with 40 μl of fresh urine from bladder of Tg-Kl and WT mice, respectively. Membrane was blotted with rat anti-Klotho antibody (KM2076). Similar results were observed in 3 animals of each group. H) Klotho protein in luminal fluid of proximal tubule obtained by in vivo free-flow micropuncture compared to bladder urine of mice. Membranes were dot-blotted with 100 nl of bladder urine from two different WT and Kl−/− mice and with 50 nl and 82 nl proximal tubule luminal fluid from WT mice, and 60 nl and 48 nl from Kl−/− mice, and blotted with KM2076 antibody. A pegylated, streptavidin-conjugated Quantum Dot 655-Sav (Quantum Dot Corp. Hayward, CA) was used as a final fluorescent detector. Calibration was performed with rMKl (Supplemental Fig. 1F).

Klotho protein induces phosphaturia in vivo

We next tested whether Klotho functions as a phosphaturic hormone in vivo. Tg-Kl mice had lower plasma phosphorus concentration compared to WT mice, while renal fractional excretion of phosphorus (FEphos) was increased (Fig. 2A) despite hypophosphatemia. To gain direct evidence that the high FEphos is proximal in origin, we performed in vitro microperfusion of microdissected single PCT and showed that Pi flux was significantly reduced in Tg-Kl compared to WT mice (Fig. 2A). NaPi-2a, a key regulator for Pi transport by the PCT (40,41,42), is slightly decreased in the kidneys of Tg-Kl mice (Supplemental Fig. 1E). We visualized two major bands of NaPi-2a (full-length and N-terminal fragment), as described previously using the N-terminal antibody (42,43,44). Full-length protein was reduced in the kidney cortex (40±7%, P<0.05, n=4) and BBMVs (52±9%, P<0.05, n=4) of Tg-Kl mice (Fig. 2B); the significance of the relative change in NaPi-2a full-length vs. N-terminal fragment will be addressed below. Since chronic transgenic elevation of Klotho can have multiple secondary effects, we examined the acute effect of Klotho on Pi homeostasis in rats. Intravenous injection of recombinant mouse extracellular domain of Klotho (rMKl; 64 pmol) induced hypophosphatemia (Fig. 3A) and increased FEphos (Fig. 3B) from 2–6 h in normal SD rats. To rule out the possibility that Klotho-induced phosphaturia was due to nonspecific proximal tubule damage, we examined and found no significant increase in urinary glucose or amino acid excretion in rats treated with Klotho (Supplemental Fig. 2AC). Therefore, phosphaturia is not a result of Klotho-induced proximal tubule injury. Rats injected intravenously with heat-inactivated Klotho did not have appreciable change in blood Pi, urinary Pi excretion and FEphos (Supplemental Fig. 2D).

Figure 2.

Figure 2.

Chronic effect of Klotho on phosphate homeostasis and NaPi-2a expression in murine kidney. A) Tg-Kl vs. WT mice. Plasma Pi (black solid circle), FEphos (red solid square) and phosphate transport (Jphos) (blue open circle) in single in vitro microperfused proximal tubules (blue open spot). n = 5; *P < 0.05, **P < 0.01 vs. WT; unpaired Student’s t test. B) Representative immunoblot of NaPi-2a and β-actin in kidney cortical membranes (cortex) and BBM from 4 WT and Tg-Kl mice/group with KM2076 and anti-β-actin. Expected mobilities of full-length NaPi-2a and NaPi-2a N-terminal are indicated.

Figure 3.

Figure 3.

Acute effect of Klotho on phosphate homeostasis and NaPi-2a expression in rat kidney. Six normal Sprague-Dawley rats were given 64 pmol of recombinant mouse Klotho (rMKl; solid line) or vehicle (Veh; dashed line). Blood and urine were collected at 0, 1, 2, 4, and 6 h. A) Plasma Pi in Klotho-treated (solid circle solid line) vs. vehicle-treated (open circle dotted line); n = 6. *P < 0.05, **P < 0.01 vs. Klotho at 0 h; #P < 0.05, ##P < 0.01 vs. vehicle at 0 h; ANOVA followed by Student-Newman-Keuls test. B) FEphos; symbols and statistical analysis same as for A. C) BBMVs were prepared, and Na+-dependent phosphate transport was measured; n = 4. *P < 0.05, **P < 0.01; unpaired Student’s t test. D) Representative immunohistochemical stain for NaPi-2a with rabbit anti-Klotho serum from 3 rats. E) Representative immunoblot for NaPi-2a and β-actin of kidney cortex membranes (cortex) and BBMVs from 3 rats/group. F) FGF23-null mice were intraperitoneally injected with 6.0 pmol of rMKl (solid line and symbols) or vehicle (dashed line and open symbols). Blood and urine were collected at 0 and 3 h and measured for plasma Pi (black) and FEphos (red); n = 4. *P < 0.05 vs. 0 h; unpaired Student’s t test.

We next measured sodium-dependent phosphate uptake in BBMVs prepared from rats injected with Klotho protein vs. vehicle. BBMV sodium-dependent phosphate uptake was clearly inhibited 2 h after Klotho injection with further reduction in 6 h (Fig. 3C). Interestingly, there was hardly any change in apical NaPi-2a staining 2–4 h after intravenous Klotho injection (Fig. 3D), but after 6 h, there was discernible reduction of NaPi-2a in BBM with increased diffuse intracellular NaPi-2a presented by immunohistochemistry (Fig. 3D). Immunoblots (Fig. 3E) showed that even though the total amount (sum of full length and N termini) of NaPi-2a protein in BBMVs did not change after 2 h, a finding apparently compatible with the immunohistochemistry in Fig. 3D, there was a clear change in the relative ratio of full-length over N-terminal NaPi-2a (96% in vehicle vs. 32% in Klotho, P<0.05; Fig. 3E). This finding will be addressed in more detail below. After 6 h, the picture is different. Total NaPi-2a was clearly reduced in BBM (121% in vehicle vs. 21% in Klotho, P<0.01; Fig. 3E, right panel) and slightly decreased in total cortex (Fig. 3E, left panel). This unequivocally indicates distinct mechanisms of regulation of NaPi-2a by Klotho after 2 or 6 h.

Because transmembrane Klotho functions as a FGF23 coreceptor and the soluble Klotho may serve weakly in this capacity (19, 20, 45), we intravenously injected rMKl into FGF23-null mice to examine whether Klotho could induce phosphaturia in the absence of FGF23. FGF23-null mice responded by lowering plasma Pi and increasing FEphos (Fig. 3F), indicating that FGF23 is not required for Klotho-induced phosphaturia and Klotho may have direct phosphaturic effects.

Klotho modulates NaPi cotransporter activity in vitro

To delineate whether there is a direct effect of Klotho on NaPi-2a, we used OK cells as an in vitro model, as it bears characteristics of the renal proximal tubule, including expression of endogenous NaPi-2a and regulation by various phosphaturic hormones (16). Direct addition of Klotho decreased sodium-dependent Pi uptake in a dose- and time-dependent manner with a half-maximum effect at ∼0.4 nM (Fig. 4A, B); and detectable change as early as 1 h of incubation at 0.4 nM (Fig. 4A, B). In OK cells transfected with NaPi-2a with eGFP tag (NaPi-2a/eGFP), Klotho did not appreciably change NaPi-2a/eGFP distribution with any consistency within 60 min (Fig. 4E), despite clearly decreased transport activity by 60 min (Fig. 4A, B). However, in contrast, NaPi-2a clearly redistributed to intracellular compartments by 2–3 h after Klotho incubation (Fig. 4E), while vehicle did not induce any internalization of NaPi-2a (Fig. 4F). These studies show that Klotho inhibits NaPi-2a transport activity in an early phase without changes in ratio of full length over N termini of NaPi-2a protein; followed by a second phase associated with internalization of NaPi-2a protein. This recapitulates the findings in the intact kidney with acute injection of Klotho.

Figure 4.

Figure 4.

Klotho effect on Na-dependent phosphate transport activity in OK cells and BBMVs and on NaPi-2a antigen in OK cells. Recombinant mouse Klotho was added to a proximal tubule-like cell line (OK cells) and BBMVs, and Na+-dependent Pi uptake was measured. A–D) Dose dependence (A) and time dependence (B) in OK cells; dose dependence (C), and time dependence (D) in BBMVs; n = 6. *P < 0.05 vs. baseline; ANOVA with Student-Newman-Keuls test. E, F) OK cells were transfected with NaPi-2a/eGFP, and live cells were monitored with fluorescent microscopy; timed images show effects of Klotho (E) and vehicle (F).

Neither mutant (8, 9) nor heat-inactivated Klotho-induced changes in NaPi-2a/GFP distribution in OK cells compared to WT Klotho (data not shown). In addition, Klotho antibodies, KM2076 (anti-Kl1) and KM2199 (anti-Kl2) (8, 9) abolished Klotho-induced internalization of NaPi-2a in OK cells, whereas incubation of mouse IgG1a/b did not abrogate Klotho effect (data not shown). Furthermore, Klotho was not able to induce endocytosis of NHE3, a membrane protein (data not shown).

To further demonstrate a direct action of Klotho on NaPi-2a, we added Klotho directly to renal BBMVs. In this in vitro cell-free system, where there is no possibility of trafficking, Klotho inhibited sodium-dependent Pi uptake in a dose- and time-dependent manner (half-maximum effect ∼1.0 nM, detectable effect ∼60 min; Fig. 4C, D). Incubation with Klotho for 2 h did not change total amount of NaPi-2a (full-length+N-terminal fragment) in BBM but did induce dramatic decrease in proportion of full-length over N-terminal NaPi-2a to 15% from 94% in vehicle incubation (Supplemental Fig. 3A). This solidifies the paradigm introduced by the data presented in the previous sections of an early inhibitory effect that does not involve NaPi-2a trafficking. These results indicate that Klotho directly inhibits NaPi-2a, even in a cell-free system with no protein trafficking.

Dependence on deglycosylation

We next addressed the possible mechanism by which Klotho decreases NaPi-2a activity. Klotho is a type I membrane protein with two conserved extracellular domains (Kl1 and Kl2) in humans (46), mice (1, 47), and rats (48) with sequence similarity with members of the glycosidase family (1, 49). Tg-Kl mice had decreased the proportion of full-length over N-terminal NaPi-2a to 5.5% BBM when compared to that in WT mice (76.1%, P<0.05) (Fig. 2B, right panel). In rats acutely injected with Klotho, there was little or no change in BBM staining of NaPi-2a (Fig. 3D) after 2 h. Compatible with this finding was the fact that apical NaPi-2a protein was minimally altered in PCT microperfused with luminal Klotho for 60 min in vitro (Supplemental Fig. 3B). After 2 h of intravenous Klotho injection, there was a clear shift of full-length to N-terminal forms of NaPi-2a in BBM, as evident by the low ratio of full-length vs. N-terminal NaPi-2a (from 96% to 32% of total), although total NaPi-2a was not significantly changed (Fig. 3E). This change was observable after 4 h (data not shown) and further accentuated after 6 h, when there was also a notable decrease in total NaPi-2a in BBM (Fig. 3E). The ∼150-kDa band of NaPi-2a in the BBM is well known (41), but its physiological role is not clear. We noted a decrease in the ∼150 kDa band in parallel with the 75 kDa with Klotho, suggesting that there is a common pathway to modulate both of them.

NaPi-2a is a highly glycosylated protein (44) with conserved asparagine-linked glycosylation sites (Supplemental Fig. 3C) (49). Incubation of BBM with PNGase F for 2 h cleaved all N-linked hybrid or high mannose oligosaccharides unless they are α(1–3) core fucosylated. This extensive deglycosylation induced a dramatic mobility shift of NaPi-2a, which is clearly different from what Klotho is inducing in NaPi-2a (Supplemental Fig. 3A). The different effect between Klotho and PNGase F on NaPi-2a mobility suggests that Klotho imparts very slight modifications on NaPi-2a.

Native NaPi-2a was not easily accessible to surface labeling, but we succeeded in obtaining a signal using maleimide-conjugated biotin in a cell overexpressing NaPi-2a to permit some degree of quantitation. Supplemental Fig. 3D shows the biotin-accessible surface fraction of transfected NaPi-2a in OK cells and Klotho reduced full-length NaPi-2a. N-terminal NaPi-2a (expected ∼50 KDa) was very faint and gave inconsistent signals, indicating that N-terminal NaPi-2a in OK cells is not biotin accessible. The N300Q+N332Q double mutant did not show any surface signal, suggesting that nonglycosylated NaPi-2a is not competent in being inserted into and/or maintain residence on the cell surface, or it may be susceptible to proteases. Fluorescent cytochemical imaging showed similar results (Supplemental Fig. 3E). Therefore, the presence of extracellular glycans is obligatory for normal localization of NaPi-2a protein in plasma membrane.

To determine whether the status of N-linked glycosylation affects NaPi cotransport activity, we directly added Klotho into cell-free BBMV prepared from rat kidney cortex, and tested whether the β-glucuronidase inhibitor, d-saccharic acid-1,4-lactone (DSAL), could block Klotho-induced inhibition of sodium-dependent Pi uptake (Fig. 5A), and the shift of NaPi-2a protein from full length toward the N-terminal fragment (Fig. 5B). While DSAL per se had no effect on baseline NaPi cotransport, it abrogated the inhibition of transport activity by both Klotho and β-glucuronidase in BBMVs (Fig. 5A). We also tested whether Klotho acts as a sialidase on NaPi-2a. In contrast, the sialidase inhibitor 2,3-didehydro-2-deoxy-N-acetyl neuraminic acid (DANA) did not affect the Klotho-induced inhibition of Pi uptake in BBMV (Fig. 6A). Similarly, in OK cells, recombinant β-glucuronidase can reproduce the effect of Klotho on NaPi cotransport activity, and both the β-glucuronidase and Klotho effects can be blocked by DSAL (Fig. 6B), but DANA did not block Klotho action (Fig. 6C). Note that Klotho shifted NaPi-2a from the full-length form to N-terminal form while total NaPi-2a (sum of full-length and N-terminal form) was not changed significantly (Supplemental Fig. 3A and Fig. 5B), and this effect could be mimicked by treatment by β-glucuronidase (Fig. 5B). Interestingly, 1.0 μM DSAL can almost completely reverse the inhibition of Pi uptake by Klotho but only modestly block the mobility shift (Fig. 5). At 10 μM of DSAL, the conversion of full-length NaPi-2a to its N terminus was blocked (Fig. 5B). The IC50 of DSAL on reversing Klotho-induced reduction of NaPi cotransport was 3.1 μM, while that for reversing mobility shift was 11.1 μM (Supplemental Fig. 3F). The dissociation of these two dose-response curves is compatible with the experiments presented below.

Figure 5.

Figure 5.

Examination of Klotho as a glucuronidase-like enzyme. A) Klotho (0.4 nM), β-glucuronidase (β-Glu) (200 μg/ml), and the β-glucuronidase inhibitor DSAL (1 μM) were added directly to BBMVs for 4 h, and Na+-dependent Pi uptake was measured. Results were summarized from 6 samples/group and analyzed by ANOVA followed by Student-Newman-Keuls test. B) Representative immunoblot of NaPi-2a and β-actin in BBMV from 3 preparations. BBMVs were treated as in A, but BBMVs were subjected to immunoblotting for NaPi-2a and β-actin. C) Representative immunoblot of NaPi-2a and β-actin in BBMV from 4 preparations. Experiment was similar to A, but protease inhibitors (PIs) were used instead of β-Glu, and 20 μg protein of BBM was subjected to immunoblot for NaPi-2a and β-actin D) Experiment was similar to C, but Na+-dependent Pi uptake was measured in BBMVs. Results were summarized from 6 independent samples/group; significant differences were analyzed by ANOVA followed by Student-Newman-Keuls test. aP < 0.05; bP < 0.01 vs. 1st bar (no Klotho, DSAL, or PIs); cP < 0.05; dP < 0.01 vs. 2nd bar (Klotho); eP < 0.05; fP < 0.01 vs. 5th bar (PIs); gP < 0.05; hP < 0.01 vs. 6th bar (Klotho+PIs).

Figure 6.

Figure 6.

Comparison of Klotho to glucuronidase and effect of inhibition of β-glucuronidase or sialidase in OK cells. A) No effect of DANA on Klotho-inhibited Na-dependent phosphate transport in BBMVs. BBMVs were incubated with recombinant Klotho protein (0.4 nM) and the sialidase inhibitor DANA (20 pM) for 4 h. Results were summarized from 6 samples/group and analyzed by ANOVA followed by Student-Newman-Keuls test. B) Klotho was directly added to OK cells. Sodium-dependent Pi uptake was measured in cells incubated with Klotho (0.4 nM), β-glucuronidase (β-Glu) (200 μg/ml), and the β-glucuronidase inhibitor DSAL (1 μM) for 4 h. Results were summarized from 6 independent samples/group. C) Dose response of the sialidase inhibitor DANA on Na+-dependent Pi uptake in OK cells incubated with DANA alone or Klotho (0.4 nM) + DANA for 4 h. While β-glucuronidase inhibition blocked the Klotho-induced inhibition, sialidase inhibition did not. Results were summarized from 6 independent samples/group.

Deglycosylated NaPi-2a is more susceptible to proteases residing in BBMVs

This is the first demonstration of the relationship between glycosylation and function for NaPi-2a (Fig. 5). This is a distinct novel mechanism, because to date, regulation of NaPi-2a activity was believed to occur exclusively via changes in protein trafficking at early phase (16) and via protein degradation at later phase (50). We addressed the relationship between deglycosylation, proteolysis, and function of NaPi-2a. We incubated BBM with Klotho, as well as DSAL and protease inhibitor cocktail. The first observation is that migrating around 30 kDa at the bottom of the gel is another N-terminal fragment, which is likely a proteolytic fragment of NaPi-2a generated in vitro (Fig. 5C). The abundance of this fragment was increased by Klotho, just like the 45-kDa N-terminal fragment.

Inhibition of deglycosylation by DSAL blocked the Klotho-induced decrease in Na+-dependent Pi transport, and Klotho-induced increase in both the 45- and 30-kDa N-terminal fragments, suggesting that deglycosylation promotes proteolysis. Incubation with protease inhibitors in vitro completely abolished the generation of the 30-kDa fragment. Next, we examined whether inhibition of proteolytic cleavege reverses Klotho-induced functional inhibition of transport. Protease inhibitors did not influence Pi transport in BBMV at baseline; neither did it abolish Klotho-induced Pi transport inhibition in vitro (Fig. 5D). This is compatible with the two differing IC50 values shown in Supplemental Fig. 3F. The uncoupling of Pi transport and proteolysis suggests that Klotho-induced deglycosylation from N-glycan is sufficient to suppress Na-dependent Pi transport and does not require proteolysis.

DISCUSSION

A great deal of attention has been devoted to Klotho as an antiaging hormone. In rodents, the phenotype of Klotho underexpression or overexpression appears to center around constellations of features of premature aging or longevity, respectively (1, 2). The study of Klotho in human biology has been limited to association studies of clinical features with Klotho polymorphisms with potential but yet undetermined significance (51,52,53,54,55,56,57,58,59). Recently, two human examples were presented where there is presumed Klotho underactivity or overactivity (6, 7). Interestingly, of the myriads of phenotypic features described in the rodent models of Klotho deficiency and excess, the human examples all displayed disturbances in mineral metabolism (6, 7), supporting the important role of Klotho in mineral homeostasis. It is important to note that the human syndromes do not quite mirror the rodent models, nor can one satisfactorily explain all of the findings in the human subjects. For example, both the low (6) and presumed high Klotho (7) states are associated with high FGF23 and hyperparathyroidism, suggesting that there is still higher level of complexity that is beyond our comprehension.

There are compelling justifications to explore the role of Klotho on mineral metabolism. Studying Klotho’s effect on phosphate metabolism is, in fact, not a departure from the antiaging effect of Klotho, as there is mounting evidence to link phosphate metabolism to aging in model organisms (19, 45).

Klotho regulates calcium homeostasis (8, 10) through renal ion transport in addition to modulation of PTH (60) and 1,25(OH)2D3 (61). The fact that hyperphosphatemia was returned to normal when Kl−/− mice were crossbred with Tg-Kl mice (1) strongly suggests Klotho maintains normal phosphate metabolism. Klotho-deficient mice exhibit increased NaPi-2a and 2c in kidney (4), indicating that hyperphosphatemia may result from renal phosphorus retention. Our in vivo data clearly reveal that Klotho induces negative phosphate balance, in part, by increasing phosphaturia. The role of the intestine, bone, and redistribution cannot be ruled out at present.

Membrane-bound Klotho functions as a coreceptor or regulator required for signaling for the potent phosphaturic factor FGF23 (19, 20, 45). Transmembrane Klotho binds to multiple FGF receptors, and the formation of Klotho/FGF23/FGFR complex is essential for transduction of FGF signaling (18). While FGF23 is phosphaturic (31, 32, 62), and its insufficiency has been implicated in renal phosphate retention (63, 64), how Klotho modulates regulation of renal phosphate handling by FGF23 is not known. Our data prove that Klotho can both function as a coreceptor for FGF23 and act by itself as a phosphaturic substance.

Klotho can function as an endocrine, paracrine, or autocrine substance. Renal DCTs and choroids plexus in brain are two major sites for expression with lower levels in other organs (1, 39, 65). The multisystemic phenotype induced by Klotho deficiency (1) indicates that Klotho works on a variety of organs. The observations that Klotho was found in mouse blood (2) and that intravenous injection of Klotho induces phosphaturia further support its endocrine actions. The facts that Klotho is expressed in the distal and proximal tubules and that these structures are intimately juxtapositioned suggest that Klotho can also function as a paracrine substance in the kidney by diffusing through the renal interstitium. The proximal tubule is a target for Klotho, and Klotho transcript and protein are expressed in the proximal tubule (Fig. 1 and Supplemental Fig. 1), rendering it a true proximal tubule autocrine system like dopamine and angiotensin II. The presence of Klotho in urine and in proximal tubule fluid further confirms this model. In addition to Klotho synthesized in situ by the proximal tubule, at present, one cannot rule out that there may be transcytosis of plasma and interstitial Klotho into the proximal lumen.

Glycosylation is the most complex post-translation modification of proteins; this process can modify signal transduction (66), cell-cell interaction (67), and membrane traffic (8, 9, 17). Transmembrane Klotho is a type I membrane protein with two extracellular tandem repeats, called Kl1 and Kl2, in humans (46), mice (1, 47), and rats (48). Kl1 and Kl2 share 20–40% amino acid sequence similarity with members of glycosidase family, including β-glucosidase, β-glucuronidase, and glycosidase (1, 49). Although the Klotho sequence is missing the critical catalytic glutamic acid residues (1), Tohyama et al. (49) identified that Klotho hydrolyzes β-glycosides and β-d-glucuronide and that its enzyme effect could be inhibited by β-glucuronidase inhibitor. Chang et al. (8) proposed that Klotho activates TRPV5 via its β-glucuronidase activity, while Cha et al. (9) showed that this activation is actually via sialidase activity. A similar Klotho effect was also found on ROMK1 (17). NaPi-2a contains N-glycosylation sites at extracellular loop 2, highly conserved among mammalian NaPi-2a proteins (44). The mutation of these two conserved asparagines to glutamines increased intracellular NaPi-2a in Xenopus oocytes (44). We found similar intracellular retention in OK cells, except in contradistinction to oocytes, cell surface NaPi-2a was drastically reduced. At the earliest time point when NaPi-2a activity is inhibited by Klotho, there is no detectable change in the total amount of NaPi-2a, but there is a direct correlation between inhibition of NaPi-2a transport and shift from the 75-kDa full-length to the ∼50-kDa N-terminal proteolytic fragment when Klotho was directly added to membrane vesicles. We did not observe notable effects of sialidase per se or its inhibitor, DANA, on NaPi-2a activity (Fig. 6) and mobility shift (data not shown), indicating that this regulation does not proceed via modulation of sialic acid. On the other hand, the glucuronidase inhibitor DSAL blocked both NaPi-2a inhibition and the increase in N-terminal fragment induced by Klotho and β-glucuronidase. It has been shown that deglycosylation could enhance membrane protein activity via an increase in surface protein (8, 9, 17) or decrease its activity through reducing surface protein (68). Protease inhibitors abolished the in vitro proteolysis of NaPi-2a completely but did not affect Klotho-induced inhibition of Pi transport activity, indicating that glycan modification without proteolysis per se is sufficient to alter transport. A number of resident proteases are found in renal BBM and are potentially involved in modulation of membrane protein and transport activity in the BBM (69,70,71). Our model predicts that glucuronate removal renders NaPi-2a protein more susceptible to proteases, but not identified yet in this study, residing in the BBM. Either the sugar modification, the proteolysis, or both processes reduce the ability of NaPi-2a to remain on the cell surface. Our findings add an additional mode of action for deglycosylation of a transporter protein, which is reduction of its intrinsic transport activity. Both regulated deglycosylation of NaPi-2a and alteration of its transport activity independent of protein trafficking and proteolysis are novel mechanisms of NaPi-2a regulation.

A summary of the proposed mode of action of Klotho on NaPi-2a is presented in Fig. 7. We have shown dependence of glucuronidase activity but have not proven that NaPi-2a is the substrate. It is plausible that Klotho deglycosylates a regulatory protein. The mechanism by which deglucuronidation reduces NaPi-2a transport activity or membrane trafficking is not known at present. In addition, Klotho may also regulate other renal sodium-dependent Pi cotransporters, NaPi-2c, (4, 11, 16), and Pit-2, (14, 15). In summary, Klotho is present in the proximal tubule apical membrane, basolateral membrane, intracellularly, as well as in the proximal urinary lumen. Klotho is a phosphaturic substance via direct actions on NaPi-2a to modulate renal phosphate excretion. Klotho modulates NaPi-2a in a biphasic fashion with dual mechanisms. It acutely (<4 h) decreases its intrinsic transport activity, and in a second phase (>4 h) induces changes in cell surface NaPi-2a (Fig. 7). The mechanism of regulation of trafficking of NaPi-2a induced by Klotho remains to be investigated.

Figure 7.

Figure 7.

Proposed model of how Klotho regulates NaPi-2a in the apical membrane of the renal proximal tubule. 1) Klotho functions acutely as a direct extracellular enzyme deglycosylating NaPi-2a protein and/or a putative regulatory protein (gray rectangle) to reduce cotransport activity. 2) NaPi-2a protein is more susceptible to resident proteases in BBM and consequently is proteolytically degraded. 3) Internalization of NaPi-2a protein from BBMVs into the intracellular pool.

Supplementary Material

Supplemental Data

Acknowledgments

This work was primarily supported by the Simmons Family Foundation. The authors also received support from the National Institutes of Health (AG19712, AG25326, DK067158, DK41612, DK-48482, DK-20543, and P30-DK-079328), the American Heart Association (0865235F), the Eisai Research Fund, the Ellison Medical Foundation, the Ted Nash Long Life Foundation, and a Seed Grant from the Charles and Jane Pak Center of Mineral Metabolism and Clinical Research. Authors are grateful to Dr. O Bonny for valuable suggestions and discussions and to Drs. J. Biber and H. Murer (Institute of Physiology, University of Zürich, Zürich, Switzerland) for antisera against rat NaPi-2a.

References

  1. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima Y I. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390:45–51. doi: 10.1038/36285. [DOI] [PubMed] [Google Scholar]
  2. Kurosu H, Yamamoto M, Clark J D, Pastor J V, Nandi A, Gurnani P, McGuinness O P, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn C R, Rosenblatt K P, Kuro-o M. Suppression of aging in mice by the hormone Klotho. Science. 2005;309:1829–1833. doi: 10.1126/science.1112766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Shiraki-Iida T, Iida A, Nabeshima Y, Anazawa H, Nishikawa S, Noda M, Kuro-o M, Nabeshima Y. Improvement of multiple pathophysiological phenotypes of klotho (kl/kl) mice by adenovirus-mediated expression of the klotho gene. J Gene Med. 2000;2:233–242. doi: 10.1002/1521-2254(200007/08)2:4<233::AID-JGM110>3.0.CO;2-5. [DOI] [PubMed] [Google Scholar]
  4. Segawa H, Yamanaka S, Ohno Y, Onitsuka A, Shiozawa K, Aranami F, Furutani J, Tomoe Y, Ito M, Kuwahata M, Tatsumi S, Imura A, Nabeshima Y, Miyamoto K-i. Correlation between hyperphosphatemia and type II Na/Pi cotransporter activity in klotho mice. Am J Physiol Renal Physiol. 2007;292:F769–F779. doi: 10.1152/ajprenal.00248.2006. [DOI] [PubMed] [Google Scholar]
  5. Lanske B, Razzaque M S. Premature aging in klotho mutant mice: cause or consequence? Ageing Res Rev. 2007;6:73–79. doi: 10.1016/j.arr.2007.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ichikawa S, Imel E A, Kreiter M L, Yu X, Mackenzie D S, Sorenson A H, Goetz R, Mohammadi M, White K E, Econs M J. A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis. J Clin Invest. 2007;117:2684–2691. doi: 10.1172/JCI31330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brownstein C A, Adler F, Nelson-Williams C, Iijima J, Li P, Imura A, Nabeshima Y, Reyes-Mugica M, Carpenter T O, Lifton R P. A translocation causing increased alpha-klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc Natl Acad Sci U S A. 2008;105:3455–3460. doi: 10.1073/pnas.0712361105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang Q, Hoefs S, van der Kemp A W, Topala C N, Bindels R J, Hoenderop J G. The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science. 2005;310:490–493. doi: 10.1126/science.1114245. [DOI] [PubMed] [Google Scholar]
  9. Cha S-K, Ortega B, Kurosu H, P. R K, Kuro-o M, Huang C-L. Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1. Proc Natl Acad Sci U S A. 2008;105:9805–9810. doi: 10.1073/pnas.0803223105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Imura A, Tsuji Y, Murata M, Maeda R, Kubota K, Iwano A, Obuse C, Togashi K, Tominaga M, Kita N, Tomiyama K, Iijima J, Nabeshima Y, Fujioka M, Asato R, Tanaka S, Kojima K, Ito J, Nozaki K, Hashimoto N, Ito T, Nishio T, Uchiyama T, Fujimori T, Nabeshima Y. alpha-Klotho as a regulator of calcium homeostasis. Science. 2007;316:1615–1618. doi: 10.1126/science.1135901. [DOI] [PubMed] [Google Scholar]
  11. Madjdpour C, Bacic D, Kaissling B, Murer H, Biber J. Segment-specific expression of sodium-phosphate cotransporters NaPi-IIa and -IIc and interacting proteins in mouse renal proximal tubules. Pflügers Arch. 2004;448:402–410. doi: 10.1007/s00424-004-1253-x. [DOI] [PubMed] [Google Scholar]
  12. Murer H, Biber J. Molecular mechanisms of renal apical Na/phosphate cotransport. Annu Rev Physiol. 1996;58:607–618. doi: 10.1146/annurev.ph.58.030196.003135. [DOI] [PubMed] [Google Scholar]
  13. Leung J C, Barac-Nieto M, Hering-Smith K, Silverstein D M. Expression of the rat renal PiT-2 phosphate transporter. Horm Metab Res. 2005;37:265–269. doi: 10.1055/s-2005-870096. [DOI] [PubMed] [Google Scholar]
  14. Villa-Bellosta R, Ravera S, Sorribas V, Stange G, Levi M, Murer H, Biber J, Forster I C. The Na+/Pi cotransporter PiT-2 (SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi. Am J Physiol Renal Physiol. 2009;296:F691–F699. doi: 10.1152/ajprenal.90623.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moe O W M. PiT-2 coming out of the pits. Am J Physiol Renal Physiol. 2009;296:F689–F690. doi: 10.1152/ajprenal.00007.2009. [DOI] [PubMed] [Google Scholar]
  16. Virkki L V, Biber J, Murer H, Forster I C. Phosphate transporters: a tale of two solute carrier families. Am J Physiol Renal Physiol. 2007;293:F643–F654. doi: 10.1152/ajprenal.00228.2007. [DOI] [PubMed] [Google Scholar]
  17. Cha S K, Hu M C, Kurosu H, Kuro-o M, Moe O, Huang C L. Regulation of renal outer medullary potassium channel and renal K+ excretion by Klotho. Mol Pharmacol. 2009;76:38–46. doi: 10.1124/mol.109.055780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goetz R, Nakada Y, Hu M C, Kurosu H, Wang L, Nakatani T, Shi M, Eliseenkova A V, Razzaque M S, Moe O W, Kuro O M, Mohammadi M. Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation. Proc Natl Acad Sci U S A. 2010;107:407–412. doi: 10.1073/pnas.0902006107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt K P, Baum M G, Schiavi S, Hu M C, Moe O W, Kuro-o M. Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem. 2006;281:6120–6123. doi: 10.1074/jbc.C500457200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444:770–774. doi: 10.1038/nature05315. [DOI] [PubMed] [Google Scholar]
  21. Nakatani T, Sarraj B, Ohnishi M, Densmore M J, Taguchi T, Goetz R, Mohammadi M, Lanske B, Razzaque M S. In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23)-mediated regulation of systemic phosphate homeostasis. FASEB J. 2009;23:433–441. doi: 10.1096/fj.08-114397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Diepens R J W, den Dekker E, Bens M, Weidema A F, Vandewalle A, Bindels R J M, Hoenderop J G J. Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport. Am J Physiol Renal Physiol. 2004;286:F483–F489. doi: 10.1152/ajprenal.00231.2003. [DOI] [PubMed] [Google Scholar]
  23. Fuster D G, Zhang J, Shi M, Bobulescu I A, Andersson S, Moe O W. Characterization of the sodium/hydrogen exchanger NHA2. J Am Soc Nephrol. 2008;19:1547–1556. doi: 10.1681/ASN.2007111245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mizuno I, Takahashi Y, Okimura Y, Kaji H, Chihara K. Upregulation of the klotho gene expression by thyroid hormone and during adipose differentiation in 3T3-L1 adipocytes. Life Sci. 2001;68:2917–2923. doi: 10.1016/s0024-3205(01)01092-x. [DOI] [PubMed] [Google Scholar]
  25. Arar M, Baum M, Biber J, Murer H, Levi M. Epidermal growth factor inhibits Na-Pi cotransport and mRNA in OK cells. Am J Physiol Renal Physiol. 1995;268:F309–F314. doi: 10.1152/ajprenal.1995.268.2.F309. [DOI] [PubMed] [Google Scholar]
  26. Sorribas V, Markovich D, Hayes G, Stange G, Forgo J, Biber J, Murer H. Cloning of a Na/Pi cotransporter from opossum kidney cells. J Biol Chem. 1994;269:6615–6621. [PubMed] [Google Scholar]
  27. Hu M C, Fan L, Crowder L A, Karim-Jimenez Z, Murer H, Moe O W. Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation. J Biol Chem. 2001;276:26906–26915. doi: 10.1074/jbc.M011338200. [DOI] [PubMed] [Google Scholar]
  28. Levine D Z. Effect of acute hypercapnia on proximal tubular water and bicarbonate reabsorption. Am J Physiol. 1971;221:1164–1170. doi: 10.1152/ajplegacy.1971.221.4.1164. [DOI] [PubMed] [Google Scholar]
  29. Schnermann J, Davis J M, Wunderlich P, Levine D Z, Horster M. Technical problems in the micropuncture determination of nephron filtration rate and their functional implications. Pflügers Arch. 1971;329:307–320. doi: 10.1007/BF00588002. [DOI] [PubMed] [Google Scholar]
  30. Yamamoto M, Clark J D, Pastor J V, Gurnani P, Nandi A, Kurosu H, Miyoshi M, Ogawa Y, Castrillon D H, Rosenblatt K P, Kuro-o M. Regulation of oxidative stress by the anti-aging hormone klotho. J Biol Chem. 2005;280:38029–38034. doi: 10.1074/jbc.M509039200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Baum M, Loleh S, Saini N, Seikaly M, Dwarakanath V, Quigley R. Correction of proximal tubule phosphate transport defect in Hyp mice in vivo and in vitro with indomethacin. Proc Natl Acad Sci U S A. 2003;100:11098–11103. doi: 10.1073/pnas.1834060100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Baum M, Schiavi S, Dwarakanath V, Quigley R. Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules. Kidney Int. 2005;68:1148–1153. doi: 10.1111/j.1523-1755.2005.00506.x. [DOI] [PubMed] [Google Scholar]
  33. Bacic D, Capuano P, Baum M, Zhang J, Stange G, Biber J, Kaissling B, Moe O W, Wagner C A, Murer H. Activation of dopamine D1-like receptors induces acute internalization of the renal Na+/phosphate cotransporter NaPi-IIa in mouse kidney and OK cells. Am J Physiol Renal Physiol. 2005;288:F740–F747. doi: 10.1152/ajprenal.00380.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Levi M, McDonald L A, Preisig P A, Alpern R J. Chronic K depletion stimulates rat renal brush-border membrane Na-citrate cotransporter. Am J Physiol Renal Physiol. 1991;261:F767–F773. doi: 10.1152/ajprenal.1991.261.5.F767. [DOI] [PubMed] [Google Scholar]
  35. Preisig P A, Ives H E, Cragoe E J, Jr, Alpern R J, Rector F C., Jr Role of the Na+/H+ antiporter in rat proximal tubule bicarbonate absorption. J Clin Invest. 1987;80:970–978. doi: 10.1172/JCI113190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Khundmiri S J, Asghar M, Banday A A, Khan F, Salim S, Levi M, Yusufi A N. Effect of ischemia reperfusion on sodium-dependent phosphate transport in renal brush border membranes. Biochim Biophys Acta. 2005;1716:19–28. doi: 10.1016/j.bbamem.2005.08.009. [DOI] [PubMed] [Google Scholar]
  37. Green J, Debby H, Lederer E, Levi M, Zajicek H K, Bick T. Evidence for a PTH-independent humoral mechanism in post-transplant hypophosphatemia and phosphaturia. Kidney Int. 2001;60:1182–1196. doi: 10.1046/j.1523-1755.2001.0600031182.x. [DOI] [PubMed] [Google Scholar]
  38. Tokuyasu K T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980;12:381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  39. Li S A, Watanabe M, Yamada H, Nagai A, Kinuta M, Takei K. Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice. Cell Struct Funct. 2004;29:91–99. doi: 10.1247/csf.29.91. [DOI] [PubMed] [Google Scholar]
  40. Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer H, Biber J. Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine. Proc Natl Acad Sci U S A. 1998;95:14564–14569. doi: 10.1073/pnas.95.24.14564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Magagnin S, Werner A, Markovich D, Sorribas V, Stange G, Biber J, Murer H. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci U S A. 1993;90:5979–5983. doi: 10.1073/pnas.90.13.5979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Biber J, Custer M, Magagnin S, Hayes G, Werner A, Lotscher M, Kaissling B, Murer H. Renal Na/Pi-cotransporters. Kidney Int. 1996;49:981–985. doi: 10.1038/ki.1996.139. [DOI] [PubMed] [Google Scholar]
  43. Xiao Y, Boyer C J, Vincent E, Dugre A, Vachon V, Potier M, Beliveau R. Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2. Biochem J. 1997;323:401–408. doi: 10.1042/bj3230401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Hayes G, Busch A, Lotscher M, Waldegger S, Lang F, Verrey F, Biber J, Murer H. Role of N-linked glycosylation in rat renal Na/Pi-cotransport. J Biol Chem. 1994;269:24143–24149. [PubMed] [Google Scholar]
  45. Razzaque M S, Sitara D, Taguchi T, St-Arnaud R, Lanske B. Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. FASEB J. 2006;20:720–722. doi: 10.1096/fj.05-5432fje. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, Nabeshima Y. Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem Biophys Res Commun. 1998;242:626–630. doi: 10.1006/bbrc.1997.8019. [DOI] [PubMed] [Google Scholar]
  47. Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida A, Anazawa H, Nagai R, Kuro-o M, Nabeshima Y. Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett. 1998;424:6–10. doi: 10.1016/s0014-5793(98)00127-6. [DOI] [PubMed] [Google Scholar]
  48. Ohyama Y, Kurabayashi M, Masuda H, Nakamura T, Aihara Y, Kaname T, Suga T, Arai M, Aizawa H, Matsumura Y, Kuro-o M, Nabeshima Y, Nagail R. Molecular cloning of rat klotho cDNA: markedly decreased expression of klotho by acute inflammatory stress. Biochem Biophys Res Commun. 1998;251:920–925. doi: 10.1006/bbrc.1998.9576. [DOI] [PubMed] [Google Scholar]
  49. Tohyama O, Imura A, Iwano A, Freund J N, Henrissat B, Fujimori T, Nabeshima Y. Klotho is a novel beta-glucuronidase capable of hydrolyzing steroid beta-glucuronides. J Biol Chem. 2004;279:9777–9784. doi: 10.1074/jbc.M312392200. [DOI] [PubMed] [Google Scholar]
  50. Pfister M F, Ruf I, Stange G, Ziegler U, Lederer E, Biber J, Murer H. Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter. Proc Natl Acad Sci U S A. 1998;95:1909–1914. doi: 10.1073/pnas.95.4.1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Friedman D J, Afkarian M, Tamez H, Bhan I, Isakova T, Wolf M, Ankers E, Ye J, Tonelli M, Zoccali C, Kuro-o M, Moe O, Karumanchi S A, Thadhani R. Klotho variants and chronic hemodialysis mortality. J Bone Miner Res. 2009;24:1847–1855. doi: 10.1359/JBMR.090516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Arking D E, Krebsova A, Macek M, Sr, Macek M, Jr, Arking A, Mian I S, Fried L, Hamosh A, Dey S, McIntosh I, Dietz H C. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci U S A. 2002;99:856–861. doi: 10.1073/pnas.022484299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Kawano K, Ogata N, Chiano M, Molloy H, Kleyn P, Spector T D, Uchida M, Hosoi T, Suzuki T, Orimo H, Inoue S, Nabeshima Y, Nakamura K, Kuro-o M, Kawaguchi H. Klotho gene polymorphisms associated with bone density of aged postmenopausal women. J Bone Miner Res. 2002;17:1744–1751. doi: 10.1359/jbmr.2002.17.10.1744. [DOI] [PubMed] [Google Scholar]
  54. Nolan V G, Baldwin C, Ma Q, Wyszynski D F, Amirault Y, Farrell J J, Bisbee A, Embury S H, Farrer L A, Steinberg M H. Association of single nucleotide polymorphisms in klotho with priapism in sickle cell anaemia. Br J Haematol. 2005;128:266–272. doi: 10.1111/j.1365-2141.2004.05295.x. [DOI] [PubMed] [Google Scholar]
  55. Yamada Y, Ando F, Niino N, Shimokata H. Association of polymorphisms of the androgen receptor and klotho genes with bone mineral density in Japanese women. J Mol Med. 2005;83:50–57. doi: 10.1007/s00109-004-0578-4. [DOI] [PubMed] [Google Scholar]
  56. Nolan V G, Adewoye A, Baldwin C, Wang L, Ma Q, Wyszynski D F, Farrell J J, Sebastiani P, Farrer L A, Steinberg M H. Sickle cell leg ulcers: associations with haemolysis and SNPs in Klotho, TEK and genes of the TGF-β/BMP pathway. Br J Haematol. 2006;133:570–578. doi: 10.1111/j.1365-2141.2006.06074.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Rhee E J, Oh K W, Yun E J, Jung C H, Lee W Y, Kim S W, Baek K H, Kang M I, Park S W. Relationship between polymorphisms G395A in promoter and C1818T in exon 4 of the KLOTHO gene with glucose metabolism and cardiovascular risk factors in Korean women. J Endocrinol Invest. 2006;29:613–618. doi: 10.1007/BF03344160. [DOI] [PubMed] [Google Scholar]
  58. Zhang F, Zhai G, Kato B S, Hart D J, Hunter D, Spector T D, Ahmadi K R. Association between KLOTHO gene and hand osteoarthritis in a female Caucasian population. Osteoarthritis Cartilage. 2007;15:624–629. doi: 10.1016/j.joca.2006.12.002. [DOI] [PubMed] [Google Scholar]
  59. Riancho J A, Valero C, Hernandez J L, Ortiz F, Zarrabeitia A, Alonso M A, Pena N, Pascual M A, Gonzalez-Macias J, Zarrabeitia M T. Association of the F352V variant of the Klotho gene with bone mineral density. Biogerontology. 2007;8:121–127. doi: 10.1007/s10522-006-9039-5. [DOI] [PubMed] [Google Scholar]
  60. Ben-Dov I Z, Galitzer H, Lavi-Moshayoff V, Goetz R, Kuro-o M, Mohammadi M, Sirkis R, Naveh-Many T, Silver J. The parathyroid is a target organ for FGF23 in rats. J Clin Invest. 2007;117:4003–4008. doi: 10.1172/JCI32409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Tsujikawa H, Kurotaki Y, Fujimori T, Fukuda K, Nabeshima Y. Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system. Mol Endocrinol. 2003;17:2393–2403. doi: 10.1210/me.2003-0048. [DOI] [PubMed] [Google Scholar]
  62. Baum M, Moe O W, Zhang J, Dwarakanath V, Quigley R. Phosphatonin washout in Hyp mice proximal tubules: evidence for posttranscriptional regulation. Am J Physiol Renal Physiol. 2005;288:F363–F370. doi: 10.1152/ajprenal.00217.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Shimada T, Kakitani M, Yamazaki Y, Hasegawa H, Takeuchi Y, Fujita T, Fukumoto S, Tomizuka K, Yamashita T. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest. 2004;113:561–568. doi: 10.1172/JCI19081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yu X, White K E. FGF23 and disorders of phosphate homeostasis. Cytokine Growth Factor Rev. 2005;16:221–232. doi: 10.1016/j.cytogfr.2005.01.002. [DOI] [PubMed] [Google Scholar]
  65. Kato Y, Arakawa E, Kinoshita S, Shirai A, Furuya A, Yamano K, Nakamura K, Iida A, Anazawa H, Koh N, Iwano A, Imura A, Fujimori T, Kuro-o M, Hanai N, Takeshige K, Nabeshima Y. Establishment of the anti-Klotho monoclonal antibodies and detection of Klotho protein in kidneys. Biochem Biophys Res Commun. 2000;267:597–602. doi: 10.1006/bbrc.1999.2009. [DOI] [PubMed] [Google Scholar]
  66. Haltiwanger R S, Stanley P. Modulation of receptor signaling by glycosylation: fringe is an O-fucose-beta1,3-N-acetylglucosaminyltransferase. Biochim Biophys Acta. 2002;1573:328–335. doi: 10.1016/s0304-4165(02)00400-2. [DOI] [PubMed] [Google Scholar]
  67. Roseman S. Reflections on glycobiology. J Biol Chem. 2001;276:41527–41542. doi: 10.1074/jbc.R100053200. [DOI] [PubMed] [Google Scholar]
  68. Ohtsubo K, Takamatsu S, Minowa M T, Yoshida A, Takeuchi M, Marth J D. Dietary and genetic control of glucose transporter 2 glycosylation promotes insulin secretion in suppressing diabetes. Cell. 2005;123:1307–1321. doi: 10.1016/j.cell.2005.09.041. [DOI] [PubMed] [Google Scholar]
  69. Girardi A C, Knauf F, Demuth H U, Aronson P S. Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells. Am J Physiol Cell Physiol. 2004;287:C1238–C1245. doi: 10.1152/ajpcell.00186.2004. [DOI] [PubMed] [Google Scholar]
  70. Ogbureke K U, Fisher L W. Renal expression of SIBLING proteins and their partner matrix metalloproteinases (MMPs) Kidney Int. 2005;68:155–166. doi: 10.1111/j.1523-1755.2005.00389.x. [DOI] [PubMed] [Google Scholar]
  71. Allred A J, Diz D I, Ferrario C M, Chappell M C. Pathways for angiotensin-(1—7) metabolism in pulmonary and renal tissues. Am J Physiol Renal Physiol. 2000;279:F841–F850. doi: 10.1152/ajprenal.2000.279.5.F841. [DOI] [PubMed] [Google Scholar]

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