Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter
Masayo Yamagata*,,
Keiichi Ozono,
Yuta Hashimoto*,
Yoshiteru Miyauchi*,,
Hiroki Kondou* and
Toshimi Michigami*
* Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health; and Department of Developmental Medicine (Pediatrics), Osaka University Graduate School of Medicine, Osaka, Japan
Address correspondence to: Dr. Toshimi Michigami, Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan. Phone: +81-725-56-1220; Fax: +81-725-57-3021; michigami{at}mch.pref.osaka.jp
Received for publication July 26, 2004.
Accepted for publication May 5, 2005.
Megalin is a multifunctional endocytic receptor that is expressedin renal proximal tubules and plays critical roles in the renaluptake of various proteins. It was hypothesized that megalin-dependentendocytosis might play a role in renal phosphate reabsorption.For addressing the short-term effects of altered megalin function,a recombinant protein for the soluble form of 39-kD receptor-associatedprotein (RAP) was administered intraperitoneally to 7-wk-oldmice. Histidine (His)-tagged soluble RAP (amino acids 39 to356) lacking the amino-terminal signal peptide and the carboxy-terminalendoplasmic reticulum retention signal was prepared by bacterialexpression (designated His-sRAP). After the direct interactionbetween His-sRAP and megalin was confirmed, mice were givena single intraperitoneal administration of His-sRAP (3.5 mg/dose).Immunostaining and Western blot analyses demonstrated the uptakeof His-sRAP and the accelerated internalization of megalin inproximal tubular cells 1 h after administration. In addition,internalization of the type II sodium/phosphate co-transporter(NaPi-II) was observed. The effects of three sequential administrationsof His-sRAP (3.5 mg/dose, three doses at 4-h intervals) thenwere examined, and increased urinary excretion of low molecularweight proteins, including vitamin D–binding protein,was found, which is consistent with findings reported for megalin-deficientmice. It is interesting that urinary excretion of phosphatewas also increased, and the protein level of NaPi-II in thebrush border membrane was decreased. Serum concentration of25-hydroxyvitamin D was decreased, whereas the plasma levelof intact parathyroid hormone was not altered by the administrationof His-sRAP. The results suggest that the His-sRAP–inducedacceleration of megalin-mediated endocytosis caused phosphaturiavia altered subcellular distribution of NaPi-II.
Megalin is a large membrane protein that belongs to the LDLreceptor–related protein family (1–3). It functionsas a multifunctional endocytic receptor together with cubilinand is expressed in the apical membrane of renal proximal tubulesand several other absorptive epithelia (4,5). Ligands that areinternalized with megalin are transported from apical clathrin-coatedpits to endosomes, where the complexes are dissociated and theligands are sorted into lysosomes to be degraded (6–8).However, megalin itself is thought to be recycled by re-fusionof endosomes and the apical membrane (6–8).
Findings in megalin-knockout mice have indicated the criticalroles of megalin in brain development and the renal uptake ofvarious filtered proteins, including vitamin D–bindingprotein (DBP), transcobalamin, and thyroglobulin (9). Althoughapproximately 98% of megalin-deficient mice die perinatallyas a result of holoprosencephaly, surviving animals exhibitincreased urinary excretion of DBP complexed with 25-hydroxyvitaminD (25OHD) and low molecular weight proteins (10–12). Urinaryloss of 25OHD results in the reduction of serum 25OHD levelsin megalin-knockout mice (11). The 1-hydroxylation of 25OHDis a critical step for vitamin D to exert its function as anactive metabolite, including maintaining the serum calcium levels,and is closely regulated by various factors, including parathyroidhormone (PTH). The 1-hydroxylation of 25OHD occurs in renalproximal tubular cells, and the uptake of 25OHD together withDBP was previously believed to be an event in the basolateralmembrane of the cells, in other words, from the bloodstream.However, megalin-knockout mice revealed that the 25OHD/DBP complexis actually filtered by glomeruli and is subsequently reabsorbedin renal tubular cells across the brush border membrane viamegalin-mediated endocytosis (11).
To further examine the function of megalin in the kidneys, kidney-specificmegalin-deficient mice have also been generated (13,14). Analysisof those mice clearly indicated the involvement of megalin inthe renal uptake of 25OHD, as well as proteins with relativelylow molecular weight. In addition, the animals exhibited impairedtrafficking of a renal Na+/Pi co-transporter (NaPi-II), suggestingthe involvement of megalin in phosphate metabolism (14).
Although it is still not clear which human disease is causeddirectly by a defect in megalin, some diseases, including Dent’sdisease and some forms of renal Fanconi syndrome, are reportedto be associated with secondary malfunction of megalin (15,16).Reduced apical expression of megalin in the proximal tubules,as well as reduced levels of megalin detected in urine, wasdemonstrated in a mouse model of Dent’s disease (17,18).In addition, some medicines such as antibiotics (aminoglycosides)and anticancer drugs exert a nephrotoxic effect and cause malfunctionof renal tubular cells (19,20). The excretion of these polybasicdrugs interferes with megalin function. For fully understandingthe roles of megalin, especially in such pathologic conditions,it is necessary to establish appropriate animal models thatallow us to elucidate the acute effects of impaired megalinfunction.
Receptor-associated protein (RAP) is a 39-kD protein that iscritical for the normal processing of megalin in various cells,including proximal tubular cells (21). RAP contains an endoplasmicreticulum (ER) retention signal at the carboxyl terminus andis mainly located in the ER (21). Indeed, RAP deficiency generatedby a gene-targeting technique is associated with reduced expressionlevels of megalin in the plasma membrane (22). When added toextracellular fluid, however, RAP binds to the ligand-bindingdomain of megalin and inhibits the binding of all of the otherligands (23). Hence, the administration of RAP to animals maymodel an acute loss of megalin function.
In this study, we hypothesized that megalin-dependent endocytosismight play a role in renal phosphate reabsorption and examinedthe short-term effects of intraperitoneal administration ofa recombinant soluble form of RAP in mice. It is interestingthat administration of soluble RAP caused phosphaturia, as wellas increased urinary excretion of low molecular weight proteins,including DBP. In addition, administration of RAP affected thesubcellular distribution of NaPi-II in proximal tubular cells.These results suggest the involvement of megalin in phosphatereabsorption in the kidneys.
Preparation of a Recombinant Soluble Form of RAP
To construct the bacterial expression vector for the solubleform of murine RAP, we performed PCR-based cloning of the cDNAusing total RNA extracted from mouse kidney. Reverse transcription(RT) was carried out using random hexamer (Promega, Madison,WI) and SuperScript II (Invitrogen, Carlsbad, CA), followedby PCR using the primers mRAP-F (sense 5'-AGAGGAAGATGGCGCCTCGAAG-3')and mRAPc-R (antisense 5'-AAGGATCCTCACCGAGCCCTTGAGACCCTGCTAGAC-3').The amplified product was cloned to pT7-Blue vector (Novagen,Madison, WI). The fragment excised by ScaI/EcoRI digestion wasthen cloned to the BamHI site in frame into the pET15b vector(Novagen) carrying an amino-terminal His-tag sequence. The resultingplasmid pET15b-sRAP encodes murine RAP corresponding to theamino acids (a.a.) 39 to 356, lacking the amino-terminal signalpeptide and the carboxy-terminal ER retention signal (HNEL asa one-letter amino acid description). The recombinant His-taggedsoluble form of RAP (His-sRAP; 342 a.a.) protein then was expressedin Escherichia coli BL21 (DE3). The expressed His-sRAP proteinwas purified using Chelating Sepharose Fast Flow (Amersham Biosciences,Piscataway, NJ). The size (approximately 40 kD) and the purityof the recombinant protein were confirmed by SDS-PAGE followedby Coomassie blue staining.
Antibodies
A polyclonal antibody was raised against megalin by preparinga recombinant protein corresponding to a 195 a.a. in the carboxy-terminalintracellular domain of mouse megalin as an antigen and immunizingwhite rabbits (16). In addition, another polyclonal antibodywas raised against a peptide representing an a.a. sequence (MMSYSERLGGPAVSP)in the amino-terminal region of NaPi-II according to a previousreport (24). The antibodies against His-tag were purchased fromSanta Cruz (His-probe; Santa Cruz, CA) and Roche (Anti-His6-Peroxidase;Mannheim, Germany). For detecting DBP, an antibody against Gc-globulinwas obtained from Dako Cytomation (Carpinteria, CA).
Ligand Blotting
Membrane fractions prepared from mouse kidneys following theprevious report (25) were subjected to SDS-PAGE and transferredto polyvinylidene difluoride membranes (BioRad, Hercules, CA).After blocking, the membrane was incubated with 0.2 mg/ml His-sRAPin a binding buffer (50 mM Tris [pH 8.0], 80 mM NaCl, 2 mM CaCl2,0.5 mM MgCl2, 1% BSA, and 0.1% Triton-X100). After washing ina washing buffer (2 mM CaCl2 and 0.5 mM MgCl2 in Tris-bufferedsaline), the membrane was immunoblotted with an anti-His6 antibodyconjugated with peroxidase (Roche). Signals were detected usingan enhanced chemiluminescence (ECL) system (Amersham Biosciences).
Animal Experiments
Animal protocols were approved by the Institutional Animal Careand Use Committee at Osaka Medical Center and Research Institutefor Maternal and Child Health. Male ICR mice (7 wk old) weresupplied by Clea Japan (Tokyo, Japan) and maintained under pathogen-freeconditions. The experimental protocols are shown in Figure 1.For examining whether intraperitoneally administered His-sRAPwas taken up by proximal tubular cells, mice were given a singleinjection of recombinant His-sRAP (3.5 mg) and then killed before(designated as 0 h) or 1, 2, or 6 h after the injection foruse in the histologic kidney analysis (Figure 1a). In anotherseries of experiments, mice were placed in metabolic cages tocollect blood and urine samples. After the collection of bloodand 20-h urine as pretreatment samples, mice were given intraperitonealadministrations of recombinant His-sRAP (3.5 mg/dose) or a vehiclethree times at 4-h intervals (Figure 1b). The 20-h urine samplesafter the second administration were collected as posttreatmenturine samples. After collection of the urine samples, the micewere killed and blood and kidneys were harvested (Figure 1b).
Figure 1. Summary of the animal experimental protocols. (a) Male ICR mice (7 wk old) were given a single intraperitoneal administration of the histidine (His)-tagged soluble form of receptor-associated protein (RAP; His-sRAP; 3.5 mg) and were killed at the indicated time points for histologic analyses of the kidneys. (b) His-sRAP (3.5 mg/dose) or PBS as a vehicle was administered intraperitoneally to 7-wk-old mice three times at 4-h intervals. Urine samples (20-h) were collected before the injections and after the second injection. Blood samples were taken before the first injection and at the end of urine sampling after the injection.
Immunostaining
The kidney specimens were fixed in 10% neutral-buffered formalin,embedded in paraffin, and cut into 5-µm-thick sections.Immunohistochemical staining was performed using the His-probe(Santa Cruz) and antibodies against megalin and NaPi-II. Whenthe antibody against NaPi-II was used, the specimens were pretreatedby incubation in 10 mM citric acid buffer (pH 6.5) at 95°Cfor 10 min. For detecting the signals, an Envision kit/HRP(DAB)(Dako Cytomation) was used.
For immunofluorescence, kidney specimens were snap-frozen inliquid nitrogen, and 4-µm-thick sections were cut. Asthe primary antibodies, His-probe raised in goat (Santa Cruz)and rabbit polyclonal antibodies against megalin and NaPi-IIwere used. As the secondary antibodies, Alexa Flour 488 donkeyanti-rabbit IgG and Alexa Flour 555 donkey anti-goat IgG wereused (Molecular Probes, Eugene, OR).
Preparation of Brush Border Membrane Fraction
The cortical brush border membrane (BBM) fraction was preparedusing a differential centrifugation method previously describedby Kumar and Prasad (26). All preparative steps were performedat 4°C. The cortices from kidneys were dissected, and a10% homogenate was prepared in ice-cold 50 mM mannitol bufferedwith 15 mM HEPES buffer (pH 7.0). MnCl2 was added to a finalconcentration of 4 mM, and the mixture was incubated at 4°Cfor 10 min. The suspension was centrifuged at 4000 x g for 15min followed by further centrifugation of the supernatant at43,000 x g for 20 min. The supernatant from this step was savedas the non-BBM fraction for subsequent Western blot analyses.The pellet was suspended and homogenized in 300 mM mannitoland 25 mM HEPES buffer (pH 6.9) and was centrifuged again at43,000 x g for 20 min. The resulting pellet was resuspendedin 300 mM mannitol and 25 mM HEPES buffer (pH 6.9). This stepwas repeated twice, and the resulting BBM pellet was resuspendedin 300 mM mannitol and 25 mM HEPES buffer (pH 6.9). Alkalinephosphatase activity was assayed to confirm the quality of theBBM fraction.
Western Blotting
The BBM and non-BBM fractions that contained 10 µg ofeach protein were subjected to SDS-PAGE and were transferredto polyvinylidene difluoride membranes (Biorad). After blocking,the membranes were incubated with the following primary antibodies:Anti-megalin antibody, His-probe, or anti–NaPi-II antibody.After incubation with the corresponding secondary antibodies,the proteins were visualized using an ECL detection system (AmershamBiosciences).
Analysis of Urine Samples
Concentrations of creatinine and phosphate in the urine sampleswere determined on an autoanalyzer. The urine samples collectedwere also subjected to SDS-PAGE on a 10% gel followed by silverstaining. For detecting DBP in urine samples, Western blot analysiswas performed using an antibody against DBP (Dako Cytomation).
Measurement of Circulating Concentrations of 25OHD and Intact PTH
The serum concentration of 25OHD was determined using a RIAkit (DiaSorin, Stillwater, MN). The plasma concentration ofintact PTH was measured using a mouse intact PTH ELISA kit (Immutopics,San Clemente, CA).
Statistical Analyses
Statistical analyses were performed using t test.
Interaction between Recombinant His-sRAP and Megalin
The purified His-sRAP was detected as a single band in SDS-PAGEfollowed by Coomassie blue staining (Figure 2a). For confirmingthe direct interaction between recombinant His-sRAP expressedin E. coli and megalin in kidney extracts, a ligand blot analysiswas performed as described in the Materials and Methods section.As a result, the antibody against His-tag detected a signalwith molecular weight comparable to that of megalin (Figure 2b).When an identical membrane was immunoblotted with the antibodyagainst megalin, a signal of similar size was detected, confirmingthe direct interaction between His-sRAP and megalin (Figure 2c).
Figure 2. Direct interaction between His-sRAP and megalin. (a) Purified recombinant His-sRAP was subjected to SDS-PAGE, followed by Coomassie blue staining. M, molecular weight marker; R, His-sRAP. Purified His-sRAP was detected as a single band. (b) Ligand blot analysis using His-sRAP. Membrane fractions that were prepared from mouse kidneys were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking, the membrane was incubated with 0.2 mg/ml His-sRAP in a binding buffer (50 mM Tris [pH 8.0], 80 mM NaCl, 2 mM CaCl2, 0.5 mM MgCl2, 1% BSA, and 0.1% Triton-X100) for 2 h at room temperature. The membrane then was washed and immunoblotted with an anti-His6 antibody conjugated with peroxidase, and signals were detected using an ECL system. The asterisk indicates the position of the signal. (c) An identical membrane to (b) was subjected to immunoblotting using an anti-megalin antibody. The asterisk indicates the position of the signal.
Uptake of Recombinant His-sRAP by Proximal Tubular Cells after Intraperitoneal Administration
The antibody against megalin immunoreacted with the BBM of theproximal tubules in untreated mice (Figure 3, 0 h). Becausethe direct interaction between His-sRAP and megalin was confirmedby the ligand blot analysis described above, we next examinedwhether intraperitoneally administered His-sRAP was taken upby proximal tubular cells. Male ICR mice were given a singleintraperitoneal injection of recombinant His-sRAP (3.5 mg/dose),then killed before (designated 0 h) or 1, 2, or 6 h after theinjection for histologic kidney analysis (Figure 1a). In immunohistochemicalexamination using the His-probe His-sRAP was detected in thesubapical area of the proximal tubule cells at time points of1 and 2 h, suggesting direct interaction between His-sRAP andmegalin expressed in the brush border (Figure 3). At the 6-htime point, His-sRAP disappeared from the proximal tubular cells(Figure 3). Injection of His-sRAP seemed to accelerate the internalizationof megalin, which was suggested by the increased dot-like stainswith anti-megalin antibody in the cytoplasm (Figure 3, arrows).
Figure 3. Immunostaining for His-sRAP, megalin, and type II sodium/phosphate co-transporter (NaPi-II) after a single intraperitoneal administration of His-sRAP. Male ICR mice were given a single intraperitoneal injection of recombinant His-sRAP (3.5 mg) and were killed before or 1, 2, or 6 h after the injection (0, 1, 2, 6 h) for immunohistochemical analysis of the kidneys using the antibodies indicated. His-sRAP was detected in the subapical area of the proximal tubular cells at the time points of 1 and 2 h, whereas it disappeared at 6 h (left). Megalin was localized predominantly in the brush border membrane (BBM) of the proximal tubular cells at 0 h, and dot-like stains were also observed in the cytoplasm at 1 h (arrows, center). NaPi-II was localized in the BBM of the proximal tubular cells at 0 h, whereas cytoplasmic staining also was observed at 2 h (arrowheads, right).
Effect of Intraperitoneal Administration of His-sRAP on Subcellular Distribution of Type II Na+/Pi Co-Transporter
Using kidney specimens obtained after a single injection ofHis-sRAP, we also performed immunostaining for NaPi-II (Figure 3).At the time point of 0 h, NaPi-II was detected in the BBMof proximal tubular cells (Figure 3). Of note, after the intraperitonealinjection, the BBM signal became weaker, and cytoplasmic stainingwas also observed (Figure 3, arrowheads).
We then performed Western blot analyses using BBM and non-BBMfractions that were obtained from kidneys after a single injectionof His-sRAP. When His-probe was used, signals correspondingto His-sRAP were detected in the samples that were obtained1 and 2 h after the injection in both the BBM and non-BBM fractions(Figure 4). Smaller signals were also detected (data not shown).When the anti-megalin antibody was used, the signal was observedin the BBM fraction at the 0-h time point. At 1 h after theinjection, megalin was detected in the non-BBM as well as theBBM fraction, which was consistent with the immunohistochemistryresults (Figures 3 and 4). At the 6-h time point, a signal wasdetected in the BBM fraction, suggesting the recycling of megalinto the apical membrane. For NaPi-II, a signal was detected inthe BBM at the time point of 0 h, whereas we observed an intensesignal in the non-BBM fraction 1 h after the injection, whichrevealed the internalization of NaPi-II (Figure 4).
Figure 4. Distribution of His-sRAP, megalin, and NaPi-II in kidney after a single intraperitoneal administration of His-sRAP analyzed by Western blot. Male ICR mice were given a single intraperitoneal injection of recombinant His-sRAP (3.5 mg) and were killed before or 1, 2, or 6 h after the injection (0, 1, 2, 6 h). The BBM fraction and non-BBM fraction were prepared as described in Materials and Methods, and 10 µg of each protein was subjected to Western blotting using the antibodies indicated.
We next investigated whether His-sRAP co-localized with megalinand/or NaPi-II in the process of endocytosis and performed doubleimmunofluorescence staining. When His-probe and the anti-megalinantibody were used, we observed the obvious co-localizationof the signals (Figure 5a). However, when His-probe and anti–NaPi-IIantibody were used, the signals overlapped only to a small extentin the apical region (Figure 5b).
Figure 5. Immunofluorescence of the kidney after the single injection of His-sRAP. Male ICR mice were given a single intraperitoneal injection of recombinant His-sRAP (3.5 mg), and the kidneys were obtained 1 or 2 h after the injection. The kidney specimens were snap-frozen in liquid nitrogen, and 4-µm-thick sections were cut on a cryomicrotome. The sections were incubated with His-probe (raised in goat) and either anti-megalin antibody (raised in rabbit) or anti–NaPi-II antibody (raised in rabbit), followed by Alexa Flour 555 donkey anti-goat IgG and Alexa Flour 488 donkey anti-rabbit IgG. (a) The distribution of megalin overlaps with that of His-sRAP in the apical region of the proximal tubular cells. (b) The distribution of NaPi-II partially overlaps with His-sRAP in the apical region.
Increased Urinary Excretion of Low Molecular Weight Proteins, DBP, and Phosphate in Mice Given His-sRAP
Mice were given three consecutive intraperitoneal injectionsof His-sRAP or a vehicle, and 20-h urine samples were collectedbefore and after the treatment as shown in Figure 1b. In theimmunohistochemical examination of kidneys that were obtained2 h after the third injection, the His-probe detected His-sRAPtaken up in the subapical area of proximal tubular cells, asfor the case of a single injection of His-sRAP (data not shown).Aliquots of 5 µl of each urine sample were subjected toSDS-PAGE followed by silver staining. The presence and increasedintensity of some signals were observed after treatment withHis-sRAP (Figure 6a). Parallel results were obtained when thesample volume was adjusted by creatinine concentration (datanot shown). Western blot analysis using the antibody againstDBP confirmed the increased urinary loss of DBP on His-sRAPadministration (Figure 6b). The urinary concentration of phosphaterelative to that of creatinine (u-Pi/Cr) was also elevated afterthree injections of His-sRAP (Figure 7). We performed immunostainingfor NaPi-II using kidneys that were obtained from these animalsand found reduced expression in the brush border in the His-sRAP–treatedmice compared with that in vehicle-treated mice (Figure 8).
Figure 6. Increased urinary excretion of low molecular weight proteins, including vitamin D–binding protein (DBP), after intraperitoneal administration of His-sRAP. Mice were given three intraperitoneal injections of His-sRAP or a vehicle, and 20-h urine samples were collected before and after the treatment as described in Materials and Methods. A 5-µl aliquot of each urine sample was subjected to SDS-PAGE followed by silver staining (a) or Western blot analysis using the antibody against DBP (b). R1 to R4, His-sRAP–injected mice; V1, V2, vehicle-injected mice; B, urine samples collected before the treatment; A, urine samples collected after the treatment. The arrow in (a) indicates the signals corresponding to albumin. The asterisks indicate bands with increased intensity after the treatment with His-sRAP.
Figure 7. Increased urinary loss of phosphate after intraperitoneal administration of His-sRAP. Mice were given three intraperitoneal administrations of His-sRAP (•; n = 9) or PBS as a vehicle (; n = 8), and urine samples were collected before and after the treatment as described in Materials and Methods. Data are expressed as fold changes in the urinary concentration of phosphate relative to that of creatinine (u-Pi/Cr) after the treatment. The circles indicate the values for individual animals.
Figure 8. Decreased amount of NaPi-II protein in the BBM after three administrations of His-sRAP. Kidney specimens were obtained from mice that were given three doses of His-sRAP (3.5 mg/dose, 4-h intervals) or PBS as a vehicle and then were subjected to immunohistochemistry for NaPi-II.
Decreased Concentration of Serum 25OHD and Unaltered Plasma PTH Levels after His-sRAP Administration
Because increased urinary loss of phosphate was observed afteradministration of His-sRAP, we determined the concentrationsof serum 25OHD and plasma PTH. Consistent with the increasedurinary loss of DBP, the concentration of serum 25OHD was significantlydecreased by His-sRAP administration (Table 1). Plasma PTH levelswere not altered by the treatment with His-sRAP (Table 2).
In this study, recombinant soluble RAP lacking the carboxyl-terminalER retention signal (His-sRAP) proved to be a useful tool toexamine the short-term effect of alteration of megalin functionwhen it was administered intraperitoneally to mice. Immunohistochemicalexamination and Western blot analyses using His-probe clearlydemonstrated the uptake of soluble His-sRAP into the proximaltubular cells, suggesting the direct effects of His-sRAP onthe functions of these cells (Figures 3 and 4). In addition,immunostaining using an antibody against megalin demonstratedthe accelerated internalization of megalin triggered by thebinding of His-sRAP (Figure 3). Western blotting also supportedthe notion that the injection of His-sRAP caused acceleratedmegalin-mediated endocytosis (Figure 4). It is interesting thatour results suggested that the stimulated megalin function wasassociated with the internalization of NaPi-II (Figures 3 and4). In addition, urinary excretion of phosphate was increasedby sequential administrations of His-sRAP (Figure 7). Phosphaturiaobserved in our model was not caused by increased secretionof PTH associated with the decrease in serum concentration of25OHD, as suggested by the unaltered plasma level of PTH afterHis-sRAP administration. Therefore, we postulated that internalizationof megalin, caused by binding of His-sRAP as the ligand, maytrigger the removal of NaPi-II from the apical membrane, leadingto increased urinary excretion of phosphate. In fact, threesequential administrations of His-sRAP resulted in a decreasedamount of NaPi-II in the proximal tubular cells (Figure 8).We assume that the internalized NaPi-II was degraded after therepeated administration of His-sRAP, although the reductionin the amount of NaPi-II protein was not obvious after a singleinjection. As previously reported, ClC-5–deficient miceexhibit secondary dysfunction of megalin and a defect in traffickingof NaPi-II (17,18). The findings in our model using His-sRAPtogether with the observation in ClC-5–deficient miceindicate the involvement of megalin in renal phosphate uptake.
In kidney-specific megalin-knockout mice, it was reported thatsteady-state levels of NaPi-II in the BBM were significantlyenhanced and that urinary excretion of phosphate was reduced(14). In addition, systemic administration of PTH resulted indefective retrieval and impaired degradation of NaPi-II in thoseanimals (14). However, in our model, wherein recombinant His-sRAPwas administered intraperitoneally, His-sRAP filtered by glomerulitriggered the accelerated internalization of megalin, and internalizationand degradation of NaPi-II associated with phosphaturia occurred(Figures 3, 7, and 8). The findings in our model are consistentwith those in the study using kidney-specific megalin-knockoutmice when the role of NaPi-II in phosphate reabsorption is considered.The difference in NaPi-II distribution and phosphate excretionbetween the two models originates from the difference in thestatus of the endocytic machinery. In our model, megalin-mediatedendocytosis is accelerated, whereas in the megalin-knockoutmouse model, the entire endocytic machinery is impaired. His-sRAPworked as an agonist rather than an antagonist for megalin assuggested by the accelerated megalin-mediated endocytosis, andit is feasible to speculate that there might be some physiologicligands for megalin that exert similar phosphaturic effects.
The mechanism by which His-sRAP–stimulated megalin-mediatedendocytosis caused the internalization of NaPi-II remains unclearat the moment. It has been reported that gentamicin, which isalso one of the ligands for megalin, causes endocytosis of NaPi-II(27), suggesting that the effects of His-sRAP are related tothe endocytic function of the liganded megalin rather than specificto His-sRAP. Using immunofluorescence, we observed co-localizationof His-sRAP with megalin, whereas His-sRAP overlapped with NaPi-IIonly to a small extent (Figure 5). Previous studies reportedthe absence of direct interaction between megalin and NaPi-II,and we also failed to detect any direct interaction in an immunoprecipitationstudy (data not shown). Other molecules might be involved inthe alteration of the subcellular distribution of NaPi-II. Themechanism for the internalization of NaPi-II might be distinctfrom the case of the Na+/H+ exchanger, whereby specific associationand co-internalization with megalin have been previously reportedin the proximal tubules (28).
Acute impairment of megalin function is often observed in renaltubulopathy caused by aminoglycosides and anticancer drugs (19,20).Hence, the administration of His-sRAP might model these conditions,and our findings suggest that urinary loss of bioactive proteins,including DBP, may require the supplementation of these substancesin such conditions. Our results suggest that acute loss of DBPin urine also may cause a decrease in serum concentration of25OHD. This finding may suggest that supplementation of 25OHDfrom the reservoir in the liver is not rapid enough to maintainits serum concentration. The effect of this transient decreasein serum concentration of 25OHD on calcium and bone metabolismis the next issue to be examined.
In conclusion, the intraperitoneal administration of His-sRAPaccelerated megalin-mediated endocytosis and caused an alterationof NaPi-II localization and phosphaturia. These results suggestthat this experimental system might provide a good model toinvestigate the relationship between phosphate reabsorptionand megalin function.
Acknowledgments
This work was supported by Grants-in-Aid from the Japan Societyfor the Promotion of Science (to M.Y., T.M., and K.O.) and grantsfrom Novo Nordisk (to T.M.) and The Mother and Child HealthFoundation (to T.M.). This work was also a part of the 21stCentury Center of Excellence entitled "Origination of FrontierBioDentistry" at Osaka University Graduate School of Dentistrysupported by the Ministry of Education, Culture, Sports, Scienceand Technology.
We thank Akihito Kimoto for technical help. We also thank TomokoHayashi and Ayami Tanaka for secretarial assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication July 26, 2004.
Accepted for publication May 5, 2005.
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Abstract
Introduction
-hydroxylation of 25OHD is a critical step for vitamin D to exert its function as an active metabolite, including maintaining the serum calcium levels, and is closely regulated by various factors, including parathyroid hormone (PTH). The 1
c-R (antisense 5'-AAGGATCCTCACCGAGCCCTTGAGACCCTGCTAGAC-3'). The amplified product was cloned to pT7-Blue vector (Novagen, Madison, WI). The fragment excised by ScaI/EcoRI digestion was then cloned to the BamHI site in frame into the pET15b vector (Novagen) carrying an amino-terminal His-tag sequence. The resulting plasmid pET15b-sRAP encodes murine RAP corresponding to the amino acids (a.a.) 39 to 356, lacking the amino-terminal signal peptide and the carboxy-terminal ER retention signal (HNEL as a one-letter amino acid description). The recombinant His-tagged soluble form of RAP (His-sRAP; 342 a.a.) protein then was expressed in Escherichia coli BL21 (DE3). The expressed His-sRAP protein was purified using Chelating Sepharose Fast Flow (Amersham Biosciences, Piscataway, NJ). The size (approximately 40 kD) and the purity of the recombinant protein were confirmed by SDS-PAGE followed by Coomassie blue staining. 
; n = 8), and urine samples were collected before and after the treatment as described in Materials and Methods. Data are expressed as fold changes in the urinary concentration of phosphate relative to that of creatinine (u-Pi/Cr) after the treatment. The circles indicate the values for individual animals.