FGFR3 and FGFR4 Do not Mediate Renal Effects of FGF23
Shiguang Liu,
Luke Vierthaler,
Wen Tang,
Jianping Zhou and
L. Darryl Quarles
Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas
Correspondence: Dr. Shiguang Liu, 3901 Rainbow Boulevard, MS 3018, Kansas City, KS 66160. Phone: 913-588-0705; Fax: 913-588-9251; E-mail: sliu{at}kumc.edu; or Dr. L. Darryl Quarles, 3901 Rainbow Boulevard, MS 3018, Kansas City, KS 66160. Phone: 913-588-9255; Fax: 913-588-9251; E-mail: dquarles{at}kumc.edu
Received for publication December 7, 2007.
Accepted for publication June 17, 2008.
Fibroblast growth factor 23 (FGF23) is a phosphaturic factorthat suppresses both sodium-dependent phosphate transport andproduction of 1,25-dihydroxyvitamin D [1,25(OH)2D] in the proximaltubule. In vitro studies suggest that FGFR3 is the physiologicallyrelevant receptor for FGF23 in the kidney, but this has notbeen established in vivo. Here, immunohistochemical analysisof the mouse kidney revealed that the proximal tubule expressesFGF receptor 3 (FGFR3) but not FGFR1, FGFR2, or FGFR4. Comparedwith wild-type mice, Hyp mice, which have elevated circulatinglevels of FGF23, exhibited low levels of serum phosphate and1,25(OH)2D, reduced expression of the sodium-dependent phosphatetransporter NPT2a in the proximal tubules, and low bone mineraldensity as a result of osteomalacia. In contrast, neither theserum phosphate nor 1,25(OH)2D levels were altered in FGFR3-nullmice. For examination of the role of FGFR3 in mediating theeffects of FGF23, Hyp mice were crossed with FGFR3-null mice;interestingly, this failed to correct the aforementioned metabolicabnormalities of Hyp mice. Ablation of FGFR4 also failed tocorrect hypophosphatemia in Hyp mice. Because the ablation ofneither FGFR3 nor FGFR4 inhibited the renal effects of excessFGF23, the kidney localization of FGFR1 was investigated. FGFR1co-localized with Klotho, the co-factor required for FGF23-dependentFGFR activation, in the distal tubule. In summary, neither FGFR3nor FGFR4 is the principal mediator of FGF23 effects in theproximal tubule, and co-localization of FGFR1 and Klotho suggeststhat the distal tubule may be an effector site of FGF23.
Fibroblast growth factor 23 (FGF23), a phosphaturic factor predominantlyproduced by osteocytes in bone,1 causes hypophosphatemia, suppressionof 1,25-dihydroxyvitamin D [1,25(OH)2D] production, and rickets/osteomalacia.1–4Excess FGF23 mediates renal phosphate wasting in hereditaryhuman hypophosphatemic disorders, including autosomal dominanthypophosphatemic rickets,5,6 X-linked hypophosphatemia,7,8 andautosomal recessive hypophosphatemic rickets.9,10 Elevated FGF23also mediates hypophosphatemia in several acquired disorders,including tumor-induced osteomalacia,11 McCune-Albright syndrome,and polyostotic fibrous dysplasia.12
FGF23 inhibits the sodium-dependent phosphate transporter andCYP27B1 activity in the proximal tubule of the kidney, leadingto phosphaturia and reduced production of 1,25(OH)2D.2,11,13,14FGF23 binds to and activates FGF receptors (FGFR) 1c, 3c, and4 in cell lines that coexpress Klotho, a transmembrane proteinco-factor that determines the tissue specificity of FGF23.15,16FGF23 has been shown to inhibit directly phosphate transportand to suppress 1-hydroxylase gene expression in isolated proximaltubules and/or proximal tubule–derived cell lines in vitro,14,17–19which were recently shown to express Klotho transcripts by reversetranscription–PCR (RT-PCR).14 Of the four members of theFGFR family, all of which are expressed in the kidney at specificlocations,20 FGFR3 is the likely physiologically relevant FGF23receptor, because it binds FGF23 in vitro and is the major FGFRexpressed in the proximal tubule,20 but the role of FGFR3 inmediating FGF23 actions has not been directly tested in vivo.To determine whether FGFR3 is the physiologically relevant FGF23receptor in the kidney, we examined sites of FGFR3 and Klothoexpression in the kidney and used mouse genetic approaches toablate FGFR3 in Hyp mice, a model of FGF23 excess characterizedby hypophosphatemia and aberrant production of 1,25(OH)2D.21–23
Localization of FGFR and Klotho in the Kidney
There are four known FGF receptors.24,25 RT-PCR analysis identifiedall four FGFR and Klotho transcripts in the adult mouse kidney(Figure 1A). By immunostaining, we observed that FGFR3 is expressedin both in the proximal tubule and the distal tubule with higherexpression in the distal tubule (Figure 1B). In contrast, FGFR1,2, and 4 were not present in the proximal tubule. Rather, FGFR1and FGFR4 were expressed in the distal tubules (Figure 1B),whereas FGFR2 was expressed mainly in the macula densa (Figure 1B).
Figure 1. Expression of FGFR and Klotho in the kidney. (A) RT-PCR analysis identifies all four FGFR (R1 through 4) and Klotho are expressed in kidney. RT-PCR products from total RNA isolated from kidney of 6-wk-old WT mice were separated and visualized in 2% agarose gel containing ethidium bromide. (B) Immunohistologic analysis of FGFR expression in the kidney. FGFR3 is expressed in the proximal and distal tubules, whereas FGFR1 and 4 are expressed in the distal tubule. FGFR2 is expressed in the macula densa. g, glomerulus; pc, proximal convoluted tubule; dc, distal convoluted tubule. The identity of the tubular segments was confirmed by immunohistochemistry with antibodies to proximal and distal markers (see Figure 7). Magnification, x200.
Effects of FGFR3 Deletion on Gross Appearance, Hypophosphatemia, and Aberrant 1,25(OH)2D Regulation in Hyp Mice
To investigate the role of FGFR3 in mediating FGF23 effects,we transferred FGFR3 deficiency onto the Hyp mouse background.Mice were genotyped by PCR (Figure 2). Mice were born at theexpected Mendelian frequency, and mutant mice had survival ratesidentical to wild-type (WT) mice. At 6 wk of age, Hyp mice displayedgrowth retardation, whereas FGFR3–/– mice showedkyphosis and wavy, elongated tails, as previously reported.26Superimposing FGFR3 deficiency on Hyp mice resulted in bothgrowth retardation and kyphosis and tail distortion in combinedFGFR3–/–/Hyp mice (Figure 2). Consistent with ourprevious findings,27 we found increased circulating FGF23 levels,hypophosphatemia, and inappropriately normal 1,25(OH)2D levelsfor the degree of hypophosphatemia in Hyp mice (Table 1) .Incontrast, the serum phosphorus, 1,25(OH)2D, and FGF23 levelsdid not differ between FGFR3-null and WT mice (Table 1). Moreimportant, in combined FGFR3–/–/Hyp mice, the absenceof FGFR3 failed to correct the hypophosphatemia or aberrant1,25(OH)2D levels in Hyp mice (Table 1). The expression of thesodium-dependent phosphate transporter (NPT2a) transcripts andprotein were decreased in the proximal tubule of both Hyp andcombined FGFR3–/–/Hyp mice (Figure 3), consistentwith the failure of FGFR3 ablation to rescue the hypophosphatemia.In contrast, the relative mRNA expression levels of Cyp27b1was slightly increased but not significantly different betweenWT and mutant mice (mean ± SEM 1.0 ± 0.1 in WT,1.5 ± 0.4 in Hyp, 1.2 ± 0.2 in FGFR3–/–,and 1.5 ± 0.4 in combined FGFR3–/–/Hyp mice;n 5), consistent with the previously reported posttranscriptionaldefect that underlies the abnormal vitamin D metabolism in Hypmice.28
Figure 2. Generation of combined Phex- and FGFR3-deficient mice. (A) Genotyping of mice by PCR. Representative PCR analysis of genomic DNA for the FGFR3 gene (top), neomycin cassette (middle), and Phex gene (bottom) in WT, Hyp, FGFR3–/–, and combined FGFR3–/–/Hyp mice. (B) Gross appearance of WT, Hyp, FGFR3–/–, and combined FGFR3–/–/Hyp mice at 6 wk of age.
Figure 3. Expression of NPT2a in kidneys from WT, Hyp, FGFR3–/–, and FGFR3–/–/Hyp mutant mice at 6 wk of age. (A) The levels of NPT2a mRNA in the kidneys from WT and mutant mice were measured by real-time RT-PCR. The values shown are means ± SEM from four samples in each group. *P < 0.05 versus WT by one-way ANOVA. (B) Immunohistologic analysis with NPT2a-specific antibody shows the expression of NPT2a in the brush border membranes of proximal tubules in kidneys from WT, Hyp, FGFR3–/–, and FGFR3–/–/Hyp mutant mice. (Insets) Higher magnification views of a single proximal tubule showing the expression of NPT2a in the brush border membranes (arrows). Hyp proximal tubules displayed a narrow band and diminished intensity of brush border membrane staining for NPT2, consistent with persistent hypophosphatemia. Magnification, x200.
Unexpected, we observed an approximately two-fold increase inboth circulating FGF23 level and FGF23 message level in boneof combined FGFR3–/–/Hyp mice above the alreadyelevated levels in Hyp mice (Table 1, Figure 4A). The FGFR3-dependentregulation of FGF23 levels was observed only in the Hyp mousebackground, and loss of FGFR3 had no effect on FGF23 levelsin mice lacking the Phex mutation. FGF23 is predominately expressedin osteocytes in cortical bone in Hyp mice.27 To explore thepossibility that FGF23 may negatively regulate its own expressionin bone through FGFR3, we examined whether the FGFR co-factorKlotho is coexpressed with FGFR3 in bone and osteoblast/osteocytecultures. We found that whereas FGFR3 was expressed in longbone, cultured osteoblasts, and, to a lesser extent, MLO-Y4osteocytes, Klotho was not found to be expressed in bone, osteoblasts,or osteocytes (Figure 4B).
Figure 4. Effect of FGF23 expression in bone by superimposed FGFR3 deficiency on Hyp mice and the expression of FGFR3 and Klotho in bone. (A) Quantitative real-time RT-PCR analysis demonstrates the relative expression of FGF23 in long bones of WT and mutant mice. Data are means ± SEM from at least three samples in each group. *P < 0.05 versus WT by one-way ANOVA; #P < 0.05 versus Hyp mice. (B) RT-PCR products revealed by ethidium bromide demonstrate the expression of FGFR3, Klotho, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in kidney and cortical bone from WT mice at 6 wk of age, TMOb osteoblasts, and MLO-Y4 osteocytes.
FGFR3 Effects on Bone Abnormalities in Hyp Mice
Hyp mice exhibited radiographic and histologic evidence of ricketsand osteomalacia. Radiographic analysis revealed reduced bonelength and bone density, widening of the growth plate, and decreasedmineralized trabecular bone in Hyp mice (Figure 5, Table 2).Bone histologic analysis showed widened growth plate, excessiveosteoid surfaces, and impaired mineralization as evidenced bydiminished and indistinct fluorescence labeling of bone in Hypmice (Figure 6). Consistent with previous reports,26,29 we foundthat FGFR3–/– mice exhibited bone dysplasia characterizedby increased bone length, curvature of the femur, and crookedtails (Figures 2 and 5, A and C). Although the bone mineraldensity was similar between FGFR3–/– and WT mice(Figure 5B), microcomputed tomography (µCT) analysis revealedwidening of the metaphyseal/epiphyseal region without changesin trabecular bone volume (Figure 5D, Table 2). Histologically,FGFR3–/– mice showed increased thickness of thehypertrophic zone, but the amount of osteoid and bone formationas assessed by fluorescence labeling was similar in FGFR3–/–and WT mice (Figure 6). Combined FGFR3–/– and Hypmice had features of both mutations. In combined FGFR3–/–and Hyp mice, the overgrowth and bending of long bone observedin FGFR3–/– mice were superimposed on the ricketsand osteomalacia of Hyp mice, and the bone mineralization defectin Hyp mice was sustained in the combined FGFR3–/–/Hypmice (Figures 5 and 6, Table 2).
Figure 5. Length and radiologic analysis of femurs from WT, Hyp, FGFR3–/–, and FGFR3–/–/Hyp mutant mice at 6 wk of age. (A) Femur length of WT and mutant mice. (B) Bone mineral density (BMD) of femurs in WT and mutant mice. (C) Three-dimensional µCT images of femurs from WT and mutant mice. Arrows indicate the curve site of femurs. (D) Three-dimensional µCT images of a longitudinal section of the distal femurs from WT and mutant mice. Data are means ± SEM from at least six samples in each group. *P < 0.05 versus WT or indicated group by one-way ANOVA.
Figure 6. Effects of superimposed FGFR3 deficiency on cartilage and bone histology. (A) Growth plate of proximal tibia of a 6-wk-old mouse (Goldner's stain). Hyp mice have a thicker growth plate as a result of increased proliferating and hypertrophic zones. FGFR3–/– mice also show an increased hypertrophic zone in the growth plate compared with WT mice but to a lesser degree than Hyp mice. The growth plate in combined homozygous FGFR3–/–/Hyp mice resembles Hyp. (B) Cortical bone of tibia of a 6-wk-old mouse (Goldner's stain). Hyp mice demonstrate impaired mineralization as evidenced by the excess osteoid in bone (red stain indicated by arrow). FGFR3–/– mice do not have notable unmineralized osteoid in cortical bone. Combined FGFR3–/–/Hyp mice retain the characteristic of mineralization defect of Hyp bone. (C) Unstained cross-section of tibias of 6-wk-old mice viewed under fluorescence light. Both WT and FGFR3–/– bones show distinct alizarin red and calcein green double labels (white arrows). Hyp bone shows diffuse and indistinct fluorescence labeling. Combined FGFR3–/–/Hyp bone is not different from Hyp bone. Magnification, x200.
Effects of FGFR3 Deletion on Hypophosphatemia, Aberrant 1,25(OH)2D Levels, and Bone Abnormalities in Hyp Mice
Because FGF23 can also bind to and activate FGFR4:Klotho complex16,18and FGFR4 and Klotho are coexpressed in the distal tubule30,31(Figure 1), we next examined the possible role of FGFR4 in mediatingthe renal effects of FGF23. This was accomplished by the transferof FGFR4 deficiency onto the Hyp mouse background. Similar toour results from FGFR3 ablation study, we found that the lossof FGFR4 had no effect on serum phosphorus or 1,25(OH)2D levelsand did not rescue the hypophosphatemia or aberrant 1,25(OH)2Dlevels in Hyp mice (Table 3). Similar to FGFR3 ablation, however,loss of FGFR4 in Hyp mice resulted in further increases in serumFGF23 levels. FGFR4-null mice displayed no bone abnormalitiesby dual x-ray absorptiometry or µCT analysis (Figure 7).In addition, ablation of FGFR4 in Hyp mice (i.e., combined FGFR4–/–and Hyp mice) failed to correct the impaired bone mineralizationdefect and rickets characteristic of Hyp mice (Figure 7).
Figure 7. Length and radiologic analysis of femurs from WT, Hyp, FGFR4–/–, and FGFR4–/–/Hyp mutant mice at 6 wk of age. (A) Femur length of WT and mutant mice. (B) BMD of femurs in WT and mutant mice. (C) Three-dimensional µCT images of a longitudinal section of the distal femurs from WT and mutant mice. Data are means ± SEM from at least 6 samples in each group. *P < 0.05 versus WT mice by one-way ANOVA.
Klotho and FGFR1 Are Co-localized in Distal Tubules
Because the ablation of neither FGFR3 nor FGFR4 in Hyp miceinhibits the renal effects of excess FGF23, we explored thelocation in the kidney of FGFR1, the remaining FGFR family membercapable of forming a complex with Klotho and FGF23. To accomplishthis, we performed immunohistologic analysis of serial kidneysections with specific antibodies to FGFR1, the proximal tubularmarker NPT2a, and the distal tubular markers Klotho and transientreceptor potential cation channel 5 (TRPV5)32,33 (Figure 8).We found that FGFR1 was co-localized in the distal tubule (Figure 8A)with Klotho and TRPV5, which where present only in the distalconvoluted tubules (Figure 8). We were unable to detect eitherFGFR1 or Klotho in the proximal tubule cells, identified bytheir expression of NPT2a (Figure 8A).
Figure 8. Expression and co-localization of FGFR1 and Klotho in the distal tubule. (A) Immunohistologic analysis of FGFR1, Klotho, and NPT2a expression in serial sections of the kidney. FGFR1 co-localizes the Klotho in the distal tubule but not NPT2a, which is expressed in the proximal tubule. (B) Immunohistologic analysis of Klotho and TRPV5 in serial sections of the kidney. Klotho co-localizes with the distal tubular marker TRPV5. Corresponding tubule segments in the serial sections are marked.
In these studies, we transferred the Hyp mouse, a model of increasedFGF23 production, onto the FGFR3-null background to evaluatethe role of FGFR3 in mediating the effects of elevated FGF23in vivo. Previous studies showed that FGF23 binds to and activatesFGFR1c, FGFR3c, and FGFR4, but not FGFR2.15,16 We found thatablation of FGFR3 did not alter serum phosphorus or 1,25(OH)2Dlevels or the expression of NPT2a in the proximal tubules inWT mice, indicating that the receptor had no demonstrable functionon systemic phosphate homeostasis or vitamin D metabolism. Moreimportant, the absence of FGFR3 in Hyp mice failed to rescuethe hypophosphatemia, aberrant 1,25(OH)2D production, or decreasedproximal tubular expression of NPT2a caused by increased circulatinglevels of FGF23 (Table 1, Figure 2).
Of the remaining FGF receptors, neither FGFR4 nor FGFR2 seemto account for the effects of FGF23 on the proximal tubule.In this regard, ablation of FGFR4, located in the distal tubule,failed to rescue the hypophosphatemia in Hyp mice (Table 3).FGFR2 is not a likely candidate for mediating FGF23 effectsin the kidney, because it does not bind to FGF23,16 and itsexpression is restricted to the macula densa. Of the remainingFGFR that are known to bind to FGF23 in the presence of Klotho,FGFR1 is the only remaining candidate. We found that FGFR1 andits co-factor, Klotho, however, are co-localized to the distaltubule of the mouse kidney (Figure 1),30,31 raising the questionof which tubular segment is the biologically relevant targetof FGF23. Use of mouse genetic approaches to evaluate the roleof FGFR1 is more difficult, because deletion of FGFR1 resultsin embryonic lethality.34,35 Thus, assessing the role of FGFR1in mediating FGF23 effects will require conditional deletionof FGFR1 from the kidney.
Although we did not rescue the hypophosphatemia by deletionof FGFR 3 and/or 4, it remains possible that these receptorsstill could participate, along with FGFR1, as redundant targetsfor FGF23 in the kidney. In support of this possibility, theobserved increase in FGF23 levels in Hyp mice lacking FGFR3and FGFR4 (Tables 1 and 3) might represent a compensatory increasein FGF23 production by bone as a result of end-organ resistancefrom the loss of FGFR3 and FGFR4. Alternatively, the incrementin FGF23 might represent a loss of feedback suppression by FGF23as a result of loss of FGFR3 or FGFR4. Although, we failed toidentify Klotho expression in bone, recent studies identifiedFGF23 effects on bone that are independent of its effects onsystemic phosphate homeostasis.36 Conversely, in combined FGFR3–/–and Hyp mice, the overgrowth and curvature of long bone observedin FGFR3 knockout mice were superimposed on the rickets andosteomalacia of Hyp mice (Figures 5 and 6), consistent withseparate roles of FGFR3 and Phex mutations in bone. Furtherstudies will be needed to investigate fully the mechanisms underlyingthe increased expression of FGF23 in bone of combined Hyp andFGFR3 and FGFR4-null mice.
Another interesting question raised by our studies is whetherthe proximal or distal tubule is the primary target for FGF23.Although we cannot exclude the possibility of low-level expressionof Klotho in the proximal tubule, this essential co-factor forFGF23 activation of FGFR is predominately present in the distaltubule, where it co-localizes with FGFR1, FGFR3, and FGFR4 (Figure 1).As an explanation for the mismatch between distal Klotho expressionand proximal tubule function of FGF23, it has been proposedthat a secreted form of Klotho may function in a paracrine/endocrinemanner to mediate the actions of FGF23 on phosphate transportand 1,25(OH)2D production in this segment of the nephron.37Alternatively, FGFR1:Klotho complex present in the distal tubulemight stimulate a distinct paracrine factor that communicateswith the proximal tubule to regulate phosphate transport and1,25(OH)2D production. Evidence for this novel distal-to-proximalfeedback mechanism includes the anatomic proximity of the distaland proximal tubule in the cortex and the observation that indomethacin,which mainly inhibits prostaglandin synthesis in tubular segmentsother than the proximal tubule,38 can correct hypophosphatemiain Hyp mice.39 Whether FGFR:Klotho complex can regulate thedistal tubular production of prostaglandins or other paracrinefactors remains to be determined. Regardless, additional studiesare needed to define the biologically relevant molecular targetsfor FGF23 in the kidney and how FGF23 regulates proximal tubularepithelial cell functions.
Finally, in contrast to the kidney, whether FGF23 has directeffect on bone is not clear. Recent in vitro studies of cultureosteoblasts demonstrated an effect exogenously of FGF23 to regulateosteoblast differentiation.36 In contrast, we failed to identifyKlotho expression in bone. Moreover, we did not identify anyattenuation of the Hyp bone phenotype associated with excessiveFGF23 levels in the combined Hyp-FGFR3 and Hyp-FGFR4–nullmice.
In conclusion, neither FGFR3 nor FGFR4 by itself accounts forFGF23 actions on the kidney. Further studies that selectivelydelete FGFR1, as well as creation of compound deletions of allthree FGFR, from the kidney may be needed to define the physiologicallyrelevant FGF23 receptors. Additional studies are needed to resolvethe paradoxic finding of expression of FGFR:Klotho complex inthe distal tubule and the proximal actions of FGF23. Knowledgeof the receptor and tubular segment that is the target for FGF23is important in understanding the actions of FGF23 on the kidneyand in developing therapeutic approaches to treat diseases causedby excess FGF23.
Transferring FGFR3 and FGFR4 Deficiency onto Phex-Deficient Hyp Background and Mouse Genotyping
FGFR3 and FGFR4 knockout mice were generated as previously reportedand provided by Dr. Weinstein (Ohio State University, Columbus,OH).40 FGFR3 and FGFR4 knockout mice are in a mixed geneticbackground consisting of 129 Sv and C57BL/B6, whereas Hyp miceare in C57BL/6 genetic background. We crossed male FGFR3-nullmice with female Hyp mice to obtain male mice heterozygous forFGFR3 (FGFR3+/–/XY) and female Hyp mice heterozygous forFGFR3 (FGFR3+/–/XHypX). We then bred the heterozygousFGFR3 males to heterozygous FGFR3/Hyp females. The mice weregenotyped by PCR (see below). Because we cannot distinguishWT and Hyp female mice by PCR, we collected data only from maleWT, Hyp, FGFR3–/– and combined FGFR3–/–/Hyplittermates in our study. We used a similar strategy to crossmale FGFR4-null mice to female Hyp mice. All mice were maintainedand used in accordance with the recommendations in the "Guidefor Care and Use of Laboratory Animals," prepared by the Instituteon Laboratory Animal Resources, National Research Council (Departmentof Health and Human Services Publication NIH 86-23, NationalAcademies Press, 1996) and the guidelines established by theUniversity of Kansas Medical Center Institutional Animal Careand Use Committee. Mice were maintained under standard dietpurchased from Harlan Talked (Madison, WI), which contains 0.6%Ca, 0.54% phosphate, and 2200 IU of vitamin D. Genomic DNA wasextracted from tail tissue of each mouse using Sigma REDExtract-N-AmpTissue PCR Kit (Sigma-Aldrich, St. Louis, MO). FGFR3- and FGFR4-nullmice and Hyp mice were genotyped using PCR methods as describedpreviously.27,40
Serum Biochemistries
Serum calcium was measured using a Calcium CPC Liquicolor Kit(Stanbio Laboratories, Boerne, TX), and serum phosphorus wasmeasured using the phosphomolybdylate-ascorbic acid method,as described previously.27 Serum parathyroid hormone levelswere measured using a Mouse Intact PTH ELISA kit (Immutopics,Carlsbad, CA). Serum 1,25(OH)2D levels were measured using a1,25 Dihydroxy Vitamin D EIA Kit (Immunodiagnostic Systems,Fountain Hills, AZ). Serum FGF23 levels were measured usingan FGF23 ELISA kit (Kainos Laboratories, Tokyo, Japan).
High-Resolution Radiography of Femurs, Bone Densitometry, and Three-Dimensional Analysis of the Femurs by µCT
The femurs from 6-wk-old mice were collected and fixed in 70%ethanol. Bone mineral densities of femurs were measured usinga PIXIMUS bone densitometer (Lunar Corp., Madison, WI). High-resolutionµCT40 (Scanco Medical, Basserdorf, Switzerland) was usedto scan and evaluate bone volume fraction and microarchitectureof the metaphyseal region of the distal femurs.23 In addition,cortical thickness data were obtained at the midshaft. The µCT40unit was calibrated weekly with a phantom standard providedby Scanco. The entire femurs were scanned in a sample holderwith 12.3-mm diameter at medium resolution, energy level of55 kV, and intensity of 145 µA. The three-dimensionalstructure was constructed and three-dimensional morphometricanalysis conducted with the built-in software of the µCTsystem. Trabecular bones from 50 cross-section slices (0.6 mm)underneath the growth plate and cortical bones from 20 cross-sectionslices at the midpoint of the femurs were analyzed using a thresholdof 250.
Cell Culture
TMOb-Nl immortalized cells derived from normal murine calvariawere grown in -minimum essential medium (Invitrogen, Carlsbad,CA) containing 10% FBS.41 The MLO-Y4, an osteocyte cell line,was provided by Dr. Lynda F. Bonewald (University of Missouriat Kansas City, Kansas City, MO). MLO-Y4 cells were maintainedin -minimum essential medium containing 5% FBS and 5% calf serumin plates coated with rat tail type I collagen as previouslyreported.42
RNA Isolation, RT-PCR, and Quantitative RT-PCR
Total RNA were extracted from homogenized kidney, bone, andcell lines using TRI Reagent (Molecular Research Center, Cincinnati,OH) and then treated with RNase-free DNase (Qiagen, Valencia,CA). First-strand cDNA was synthesized using iScript cDNA SynthesisKit (Bio-Rad, Hercules, CA). Total RNA (1 µg) was usedin each 20-µl reverse transcriptase reaction. For RT-PCR,1 µg of RNA was used in each PCR reaction. The RT-PCRproducts were separated and visualized in 2% agarose gel containingethidium bromide. For real-time RT-PCR, 200 ng of total RNAwas used in each PCR reaction. The iCycler iQ Real-Time PCRDetection System and iQ SYBR Green Supermix (Bio-Rad) were usedfor real-time quantitative PCR analysis. The relative gene expressionwas expressed as described previously using cycle threshold(Ct) values of the gene of interest normalized with cyclophilinA in the same sample.27 Sequences of primers used for regularand real-time quantitative RT-PCR are listed in Table 4.
Immunohistochemistry and Immunohistofluorescence
Kidneys collected from WT and mutant mice were fixed in 4% paraformaldehydein PBS (pH 7.4) and then embedded either in paraffin or in frozenembedding medium (Thermo Shandon, Waltham, MA). Five-micrometerparaffin sections were used for immunohistochemistry. Briefly,sections were dewaxed, rehydrated, and incubated with TargetRetrieval Solution (Dako Corp., Carpinteria, CA) at 95°Cfor 30 min. The sections were then treated with 3% H2O2 in methanoland blocked with the Avidin and Biotin Block Solution (VectorLaboratories, Burlingame, CA) and with diluted blocking serumfrom the species in which the secondary antibody was made. Then,primary antibodies were incubated on the section at 4°Covernight and detected by diluted biotinylated secondary antibodiesfollowed by incubation with the Vector ABC Reagent (Vector Laboratories).Finally, the slides were developed with diaminobenzidine substrate(Vector Laboratories) and counterstained with Mayer's hematoxylin.For immunofluorescence, 5-µm cryosections were used. Briefly,sections were rinsed with TBS and blocked with TBS containing10% normal serum from same species and 1% BSA for 1 h at roomtemperature. Then sections were incubated with primary antibodiesovernight at 4°C and detected by diluted Alexa Fluor dye-labeledsecondary antibodies. Finally, the sections were washed withTBS and mounted with mounting solution. Rabbit-raised anti-humanFGFR1 through 4 antibodies and TRPV5 antibody were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA). The negativecontrol sections in this study were incubated with nonimmunizedserum or 5 µg/ml blocking peptides (Santa Cruz Biotechnology).Rabbit-raised anti-NPT2a antibody was a gift from Dr. MosheLevi (University of Colorado Health Sciences Center, Denver,CO). The monoclonal rat anti-Klotho antibody (KM2076) was providedby Kyowa Hakko Kogyo Co. Ltd.30 The Alexa Fluor 488–labeledsecondary antibody (Invitrogen) was used in the immunohistofluorescencestaining.
Histologic Analysis of Nondecalcified Bone
Mice were prelabeled at 6 and 1 d before they were killed fortissue collection with alizarin complexone (Acros Organics,Fair Lawn, NJ) and calcein (Sigma-Aldrich), respectively, byintraperitoneal injection. Bones were fixed in 70% ethanol andembedded in methyl methacrylate. Five-micrometer sections werestained with Goldner's stain and analyzed under transmittedlight, and 10-µm unstained sections were evaluated underfluorescence light.27
Statistical Analysis
We evaluated differences between groups by one-way ANOVA formultiple-group comparison and t test for two-group comparison.All values are expressed as means ± SEM. P < 0.05was considered statistically significant. All computations wereperformed using the GraphPad Prism 4 software (GraphPad Software,San Diego, CA).
This project was supported by National Institutes of Healthgrant RO1-AR45955 from the National Institute of Arthritis andMusculoskeletal and Skin Diseases.
We thank Dr. Michael Weinstein (Ohio State University), Dr.Moshe Levi (University of Colorado Health Sciences Center),Dr. Lynda F. Bonewald (University of Missouri at Kansas City),and Kyowa Hakko Kogyo Co. Ltd. for kindly providing the FGFR3knockout mice, NPT2a polyclonal antibody, MLO-Y4 cell line,and Klotho antibody, respectively.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
-hydroxylase gene expression in isolated proximal tubules and/or proximal tubule–derived cell lines in vitro,14,17–19 which were recently shown to express Klotho transcripts by reverse transcription–PCR (RT-PCR).14 Of the four members of the FGFR family, all of which are expressed in the kidney at specific locations,20 FGFR3 is the likely physiologically relevant FGF23 receptor, because it binds FGF23 in vitro and is the major FGFR expressed in the proximal tubule,20 but the role of FGFR3 in mediating FGF23 actions has not been directly tested in vivo. To determine whether FGFR3 is the physiologically relevant FGF23 receptor in the kidney, we examined sites of FGFR3 and Klotho expression in the kidney and used mouse genetic approaches to ablate FGFR3 in Hyp mice, a model of FGF23 excess characterized by hypophosphatemia and aberrant production of 1,25(OH)2D.21–23 
5), consistent with the previously reported posttranscriptional defect that underlies the abnormal vitamin D metabolism in Hyp mice.28 
