Biochemical and Biophysical Research Communications 267, 597– 602 (2000)
doi:10.1006/bbrc.1999.2009, available online at http://www.idealibrary.com on

Establishment of the Anti-Klotho Monoclonal Antibodies
and Detection of Klotho Protein in Kidneys
Yukinari Kato,* Emi Arakawa,* , † Satoko Kinoshita,* , § Akio Shirai,* Akiko Furuya,*
Kazuya Yamano,* Kazuyasu Nakamura,* Akihiro Iida,* Hideharu Anazawa,* Noritoshi Koh,§
Akiko Iwano,§ Akihiro Imura,§ Toshihiko Fujimori,§ Makoto Kuro-o,† ,‡ Nobuo Hanai,*Kazuhiko Takeshige,* and Yo-ichi Nabeshima† ,§ ,1*Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194-8533, Japan;†Division of Molecular Genetics, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira,Tokyo 187-0031, Japan; ‡Department of Pathology, University of Texas Southwestern Medical Center
at Dallas, Dallas, Texas 75235-9072; and §Department of Pathology and Tumor Biology
Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Received December 2, 1999

A novel gene, klotho (kl), which is involved in thedevelopment of a syndrome resembling human aging
in mice, was recently identified. The kl gene encodesa single-pass membrane protein whose extracellulardomain carries homology to ␤-glucosidases. Therealso exists a splice variant of kl mRNA which encodes a putative secreted protein in both human andmouse. In this study, to characterize the physiological roles of Klotho protein, we established three
monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named
KM2076 (rat IgG 2 a), KM2119 (rat IgG 2 b), and KM2365(mouse IgG 1 ). In Western blots, KM2076 and KM2119
specifically recognized a 130 kDa Klotho protein in
the mouse and human kidney membrane fractions.
To detect the human Klotho protein, the sandwichtype ELISA system with KM2076 and KM2365 wasestablished. Using the ELISA system, we detected
the human Klotho protein as low as 20 ng/ml in the
supernatant of Chinese hamster ovary cells (CHO
cells), introduced the human klotho gene. KM2076
and KM2119 specifically gave a positive staining byimmunohistochemical staining in paraffin or frozen
sections of the kidneys from wild-type mice but notin those from kl mice. Strong staining was observed
especially in cortical renal tubules of the mouse kidney, where expression of klotho transcripts overlaps. KM2076 also showed a similar reaction patternin the paraffin sections of rat and human kidneys.
Abbreviations used: mAb, monoclonal antibody; PBS, phosphatebuffered saline; ELISA, enzyme-linked immunosorbent assay; RTPCR, polymerase chain reaction with reverse transcription; ABTS,
2,2⬘-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).
1
To whom correspondence should be addressed. Fax: 81-75-7534683. E-mail: nabemr@lmls.med.kyoto-u.ac.jp.

The mAbs established in this paper will serve as
useful analytical, pathological, and diagnostic tools
to disclose the role of Klotho protein in the suppression of a syndrome resembling human aging. © 2000
Academic Press

A novel mouse model for human aging was recently
established and the gene responsible for it was identified (1). A decrease in the expression of a novel geneklotho (kl) in mice, leads to a syndrome resemblinghuman aging. These mice, klotho mice, exhibited multiple disorders, such as arteriosclerosis, osteoporosis,
skin atrophy, pulmonary emphysema, short life-span
and infertility. The kl gene encodes a novel single passmembrane protein (1,014 a.a.) consists of an
N-terminal signal sequence, a putative extracellulardomain with two internal repeats (hKL1 and hKL2), asingle membrane-spanning region, and a short intracellular domain. These internal repeats share the sequence similarity to the ␤-glucosidase enzymes (2, 3).
The human homologue of the kl gene encoding a membrane protein (1,012 a.a.) was also isolated (4). Fromthe cDNA analysis, it was found that there exists a
splice variant of kl mRNA which encodes a putative
secreted protein (human: 549 a.a., mouse: 550 a.a.)lacking the second internal repeat of the extracellulardomain (hKL2), the transmembrane domain and theintracellular domain (4, 5). Expression of the mouse kl
gene was detected only in kidney and brain by Northern blot analysis. But the lower expression was observed in the pituitary gland, placenta, skeletal muscle, urinary bladder, aorta, pancreas, testis, ovary,colon and thyroid gland by RT-PCR (1). On the other597

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hand, the human kl gene transcript was expressed
predominantly in the kidney, placenta, prostate andsmall intestine (4).
Recent studies showed that the pathophysiological significance of the Klotho protein in some rat models forhuman diseases such as (a) spontaneously hypertensive
rat, (b) deoxycorticosterone acetate-salt hypertensive rat,(c) 5/6 nephrectomized rat, (d) non-insulin-dependent
diabetes mellitus rat models (6). The expression levels ofkl mRNA in the kidney in these rat models were significantly lower than controls, indicating that the expressionof the kl gene in the kidney is regulated under sustained
circulatory stress such as a long-term hypertension, diabetes mellitus, and chronic failure.
In this study, we immunized rats and mice with thehuman recombinant Klotho protein and establishedmAbs to detect Klotho protein in the western blots,
ELISA system and immunohistochemical staining.
These mAbs will serve as useful analytical, pathological and diagnostic tools to disclose the role of Klotho
protein in the suppression of a syndrome resemblinghuman aging.
MATERIALS AND METHODSSpecimens. Paraffin-embedded blocks of human tissues werekindly provided by Dr. T. Kamei of Yamaguchi Prefectural Central
Hospital (Yamaguchi, Japan). Freshly isolated tissues from mice andrats were immediately fixed in 10% buffered formalin.
Expression of human recombinant Klotho protein. A SacII cDNA
fragment which encodes a part of hKL1 (amino acids 55–261) and acDNA fragment (KpnI-ApaI) which encodes a part of hKL2 (aminoacids 801–954) were subcloned into a vector derived from pGHA2 (7).
The recombinant protein was expressed in Escherichia coli in inclusion bodies and was purified from SDS-polyacrylamide gel.
Hybridoma production and purification of mAbs. Rat monoclonal
antibody (mAb), KM2076, was established by immunizing rats witha part of hKL1 (amino acids 55–261), and that of KM2119 wasestablished by immunizing rats with a part of hKL2 (amino acids
801–954). KM2365 was established by immunizing mice with a synthesized amino peptide (DDAKYMYYLKKFIMETLKAIKLDGV),
which is a part of the human Klotho protein (8). The spleen cells werefused with mouse myeloma P3U1 cells, and the culture supernatantsof the hybridomas were screened by ELISA for their ability to recognize the human recombinant Klotho protein. Hybridoma cells were
cultured in RPMI growth medium, harvested by a brief centrifugation, and suspended in PBS. Four-week-old female BALB/c nude
mice (CLEA Co., Tokyo, Japan) were given i.p. injections of thehybridoma cells. The mice were sacrificed 7 days later, and ascites
fluid was collected. The mAbs were purified using protein
G-Sepharose 4B gel beads (Zymed, San Francisco, CA).
Western blot analysis. Kidneys obtained either from human orfrom mice of 5-week-old wild-type or klotho mice were homogenizedin the homogenize buffer (20 mM Tris-HCl at pH 7.5, 0.25 M Sucrose,
1 mM EDTA, 10 mM EGTA, 10 mM 2-mercaptoethanol) by 6 passes
in a Tefronglass homogenizer. Each homogenate was centrifuged at
100,000 ⫻ g at 4°C for 10 min. The supernatant was centrifugedagain at 100,000 ⫻ g at 4°C for 1 h. After the pellet was resuspended
in homogenize buffer, membrane proteins were extracted with 1%
Triton X-100. Samples from mice kidneys (20 ␮g) or from humankidneys (100 ␮g) were boiled for 5 min, with 1:4 dilution of thesample buffer containing 200 mM Tris-HCl (pH 6.8), 4% SDS, 100

mM DTT, 40% glycerol, and 0.04% bromophenol blue. Proteins in thesamples were separated on SDS-PAGE gel (7.5–15% gradient) andtransferred onto PVDF membranes. The membranes were blockedwith 5% skim milk in PBS and were incubated with the culture
supernatant at 37°C for 1 h. They were washed and were incubatedwith peroxidase-conjugated anti-rat IgG antibody (Amersham) at
37°C for 1 h. For visualizing bands, the ECL Western blotting detection system (Amersham) was used.
Biotinylation of antibodies. Biotinylation of KM2365 was performed with Antibody Biotinylation Kit (American Qualex, San
Clemente, CA). Briefly, a purified KM2365 mAb (5 mg) was dissolved
in 0.6 ml of carbonate buffer concentrate. Six hundred microliters of
biotin solution (1 mg/ml) was added into the antibody solution andgently shook for 1 h at room temperature. Unreacted biotin wasremoved by passing through G25 column. The biotinylated KM2365
was stabilized by adding BSA to a final concentration of 1% (w/v) andstored at 4°C until use.
Sandwich ELISA for the detection of Klotho protein. Klotho protein level in the supernatant was determined with the sandwich
ELISA as follows. Fifty microliters of KM2076 (10 ␮g/ml) in PBS wasadded into each well of the 96-well ELISA plate and incubatedovernight at 4°C, and the wells were washed with PBS for 3 times
and flooded with 1% BSA-PBS for 1 h at room temperature. The
secreted isoform of human Klotho protein was constitutively expressed using CHO cells (unpublished data) and the supernatant (40␮l) was diluted and applied into each well and incubated overnight at
4°C. The supernatants were then aspirated and the wells were
washed with 0.05% Tween 20 in PBS for 5 times. Fifty microliters of
biotinylated KM2365 (10 ␮g/ml) in 1% BSA-PBS were added andincubated for 2 h at room temperature. After the wells were thoroughly washed with 0.05% Tween 20-PBS for 5 times, 50 ␮l ofavidin-biotin-peroxidase complex (Vectastain DAB kit: Vector Laboratories Inc., Burlingame, CA) were added and incubated for 1 h atroom temperature. After washed with 0.05% Tween 20-PBS for 5
times, 100 ␮l of ABTS solution with 0.1% H 2O 2 were applied for 10
min at room temperature, and the absorbance at 415 nm and 490 nm
was measured.
In situ hybridization. 35S-Labeled riboprobes were prepared fromthe full-length cDNA for mouse klotho (1). Antisense and sense[ 35S]cRNAs were synthesized from linearlized plasmids in the presence of [ 35S]UTP (1289 Ci/mmol; New England Nuclear Corp; Boston,MA). In situ hybridization was performed as described previously (9).
In brief, sections were postfixed with 4% PFA-PBS for 15 min. Afterwashing with PBS, sections were digested with 1 ␮g/ml proteinase K(37°C for 15 min) in PBS and again treated with 4% PFA-PBS (10min). Sections were then sequentially washed with PBS, incubatedwith 0.2 N HCl (10 min), again washed with PBS, acetylated with
0.25% acetic anhydride in the presence of triethanolamine (0.1 M, 10min), dehydrated with increasing concentrations of ethanol, and air
dried. Hybridization with 35S-labeled complementary RNAs (cRNAs;ca. 5 ⫻ 10 7 cpm/ml) were performed in a humidified chamber in asolution containing 50% formamide, 10% dextran sulfate, 1⫻ Denhardt’s solution, 600 mM NaCl, 10 mM Tris-HCl (pH 7.6), 1 mM
EDTA, 50 mM dithiothreitol, 0.25% sodium dodecyl sulfate, and 200
␮g/ml transfer RNA (18 h; 55°C).
After hybridization, sections were washed briefly with 5⫻ SSC(20⫻ SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0) at 50°C, 50%
formamide–2⫻ SSC (50°C for 30 min), and 10 mM Tris-HCl (pH7.6)-500 mM-1 mM EDTA (TNE; 37°C; 10 min). Sections were thentreated with 10 ␮g/ml ribonuclease-A in TNE (37°C; 30 min). Afterbeing washed with TNE, sections were incubated once with 2⫻ SSC(50°C; 20 min) and twice with 0.2⫻ SSC (50°C; 20 min), dehydrated
with increasing concentrations of ethanol, and air-dried. Slides werethen placed on X-ray films (Fujifilm, Tokyo, Japan) and film autoradiographs were obtained after overnight exposure. Slides weredipped into NTB-2 (Eastman Kodak, New Haven, CT) and stored at
4°C for specific times estimated from the intensity of the expression598

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on the X-ray film. After development, sections were counterstainedwith hematoxylin and eosin and mounted.
Immunoperoxidase staining (paraffin sections). Paraffin sections
were dewaxed in xylene, hydrated through graded alcohol solutions
and washed with PBS. Endogenous peroxidase was blocked by immersion in 0.3% (wt/vol) hydrogen peroxide in absolute methanol for
30 min at room temperature. The following steps were carried out
using a Vectastain ABC kit. Briefly, after washed with PBS for 5
times, nonspecific binding was blocked with VECTASTAIN RABBIT
Normal Serum for 20 min at room temperature. Then KM2076 (10␮g/ml) were applied on sections and incubated for 1 h at room
temperature. Then sections were washed with PBS (5 times) andwere incubated with anti RAT IgG. DAB substrate was diluted to thehalf and applied. Sections were incubated 2 to 4 min according to thetime predetermined and the reaction was terminated by immersing
sections into deionized water for 5 min. Sections were counterstained
either with hematoxylin or with methyl-green, and dehydrated withincrease concentration of ethanol solution and then with xylene andmounted.
Immunoperoxidase staining (frozen sections). Mice were either
anesthetized with ether or killed by rapid cervical dislocation. Kidneys were removed, cut into slices in 4% paraformaldehyde (PFA)/PBS and further fixed in the same solution overnight at 4°C. They
were rinsed sequentially with PBS containing 5 and 10% sucrose for
4 h and then with the same buffer containing 15% sucrose overnightat 4°C. Tissues were placed in O.C.T. compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and frozen with liquid nitrogen. Frozen
sections (6 ␮m thickness) were prepared and mounted on silanecoated glass slides. To block the endogenous peroxidase, sections
were first washed 3 times in cold 10% sucrose/PBS solution (5 mineach) and then incubated in 0.3% H 2O 2 in methanol for 15 min atroom temperature and rinsed off with PBS. Nonspecific binding was
blocked by incubating sections with 10% nonimmune goat serum(Zymed Laboratories, Inc., CA) for 10 min at room temperature.
KM2076 (2 ␮g/ml in 1% BSA/PBS) was applied on sections and they
were incubated overnight at 4°C. After rinsed with cold 10% sucrose/
PBS three times, sections were incubated with peroxidaseconjugated anti body (goat anti-rat IgG(H ⫹ L) HRP, Chemicon
International Inc.) for 4 h at room temperature. After washed withcold 10% sucrose/PBS three times, DAB solution was diluted to thehalf and was applied for 1 min at room temperature as described inthe Vectastain DAB kit protocol. Sections were washed and stainedwith hematoxylin.
Fluorescence staining (frozen sections). Sections were prepared
as described in immunoperoxidase staining (frozen sections) except

FIG. 1. Immunoblot analysis of the Klotho protein in the kidneys. Immunoblot analysis of the Klotho protein in the kidneys ofwild (lane 1) and kl mice (lane 2) with (A) KM2076 and (B) KM2119.
Immunoblot analysis of the Klotho protein in the human kidneys(lane 1) with (C) KM2076 and (D) KM2119.

FIG. 2. Standard curve of sandwich ELISA for the detection of
the Klotho protein.

for using avertin (0.8 ml/head) as anesthetic. After washing out
O.C.T. compound with PBS, nonspecific binding was blocked with
10% goat serum for 15 min. KM2119 (ascites, 100⫻ dilution with 3%
FCS-PBS) incubation were done for 1 h followed by washing with
PBS. Sections were incubated with Alexa Fluor 488 goat anti-rat
IgG(H ⫹ L) conjugate (200⫻ dilution with 3% FCS-PBS, Molecular
Probes, Inc., OR) for 30 min (dark) and washed with PBS. Samples
were observed with a Leica TCS SP confocal laser scanning microscope (absorbance 488 nm and emission 520 nm).

RESULTS AND DISCUSSION
Establishment of anti-human Klotho mAbs. Here,
we presented the establishment of a mAbs against
Klotho protein. We immunized rats and mice with therecombinant human Klotho protein or a synthesizedamino peptide and obtained three mAbs (KM2076,
KM2119 and KM2365) as described in Materials andMethods (8). Matsumura et al. (4) reported that there
are two transcripts which apparently arise from alternative splicing of kl mRNA. One transcript encodes asingle-pass membrane protein with internal repeats(hKL1, hKL2). The other transcript encodes only hKL1
domain corresponding to a secreted-form of Klotho protein. As the immunized antigen for KM2119 was a partof hKL2 domain, this antibody does not recognize thesecreted form. On the other hand, the immunized antigen for KM2076 and KM2365 was a part of hKL1,
these mAbs could react with both the membrane andthe secreted form of Klotho protein.
Typical results of western blots were shown in Fig. 1.
KM2076 specifically recognized a 130 kDa Klotho protein exists in the membrane fraction from the wild-type
mouse kidney, but not that from the kl mice (Fig. 1A).
KM2076 also recognized specifically a 130 kDa Klotho
protein in the membrane fraction of human kidney(Fig. 1C). KM2119 also recognized the 130 kDa Klotho
protein in the membrane fraction from the wild-type
mouse kidney (Fig. 1B) and the human kidney (Fig.
1D), but not that from the kl mice (Fig. 1B). Interestingly, KM2119 additionally recognized a 60 kDa protein as well as a 130 kDa one in the wild-type mouse599

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FIG. 4. Immunoperoxidase staining of kidneys with KM2076. Paraffin sections of (A and B) rat or (C and D) human kidneys. (A and C)
KM2076 and the secondary mAb were sequentially applied as described under Materials and Methods. (B and D) The secondary antibody
but not KM2076 was applied. Sections were counterstained with hematoxylin. Bar represents 100 ␮m.

(Fig. 1B) and the human (Fig. 1D) kidneys. It might bepossible that a part of extracellular domain of Klotho
protein was cleaved and the rest of Klotho protein nearKL2 domain was recognized as 60 kDa protein.
Sandwich ELISA for the detection of Klotho. The
supernatant of CHO cells expressing human klotho
cDNA was serially diluted, and the level of Klotho
protein was determined with the sandwich ELISA. As
shown in Fig. 2, the value of A415 corresponded well tothe concentration of Klotho protein. Using the ELISA,
we concluded that the Klotho protein as low as theconcentration of 10 to 20 ng/ml could be detected.
Localization of the klotho transcript and Klotho protein. Strong signals of klotho transcripts were observed on the cortical renal tubules of the wild-type

mouse kidney, but not those of the kl mouse (Fig. 3A,
B). The interesting point is that not all the cells in thecortical renal tubules express the mRNA. These signals are specific for kl transcripts, because we did notdetect any signals with the kl sense probes (data notshown). The pattern of localization of kl transcripts
was similar to that reported earlier (1).
To select mAbs available for the immunohistochemical detection of Klotho protein, we first stained kidneys from wild-type and kl mice. Among mAbs tested,
KM2076 specifically stained the cortical renal tubulesof wild-type mice kidney in both frozen (Fig. 3C) andparaffin sections (Fig. 3E) by immunoperoxidase staining. We concluded that the staining was specific for
Klotho protein, because we did not detect any staining

FIG. 3. Expression of klotho mRNA and Klotho protein in the mouse kidneys. (A and B) Expression of mRNA for kl in the kidney of
7-week-old mouse. In situ hybridization was performed on sections with 35S-labeled cRNA probes that was specific for mRNA for kl. (A)Bright-field image with the kidney from the wild-type mouse. Expression of kl mRNA was detected as the dense localization of black grainson the renal tubules. (B) Bright-field image with the kidney from kl mice. (C and D) Immunoperoxidase staining of kidneys with KM2076.
Frozen sections of kidneys isolated from (C) wild-type or (D) kl mice were stained with KM2076. Sections were counterstained withhematoxylin. (E and F) Immunoperoxidase staining of kidneys with KM2076. Paraffin sections of kidneys isolated from (E) wild-type or (F)kl mice were stained with KM2076. Sections were counterstained with methyl-green. (G) Fluorescence staining of the wild-type mouse kidneywith KM2119. Green signals represent signals from Klotho staining. The bars under B, F, and G represent 100 ␮m for A, B, E, F, and G, andthe bar under D represents 50 ␮m for C and D.
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on the cortical renal tubules of kl mice kidney (Figs. 3Dand 3F). KM2119 also specifically stained the cortical
renal tubules of wild-type mice kidney on frozen sections by fluorescence staining (Fig. 3G). The localization of Klotho protein in the mouse kidney was similar
to that of kl transcripts. Using KM2076, we further
stained the paraffin sections of the rat (Figs. 4A and4B) and the human (Figs. 4C and 4D) kidneys. KM2076
also gave specific staining on the cortical renal tubulesof rat (Fig. 4A) and human (Fig. 4C) kidneys.
In conclusion, among the monoclonal antibodies established, KM2076 can be used to detect human Klotho
protein for the Western blot analyses. Also KM2076
can be used to monitor the secreted form of human
Klotho in ELISA system with the combination with
KM2365. KM2119, which detects the KL2 part ofKlotho, gave an additional 60 kDa band as well as 130
kDa band in the samples from human and mouse kidneys in Western blot. For immunohistochemical analysis, KM2076 gave a specific staining of Klotho inmouse, rat, and human kidney sections. KM2119 also
gave a similar result in mouse kidney sections by using
fluorescence staining. Besides the characterization
above, we also have a preliminary data of the availability of KM2076 and KM2119 for the immunoprecipitation of Klotho protein.
These antibodies will be powerful tools for understanding the function of Klotho.

ACKNOWLEDGMENTS
We thank Hiroe Sato, Yukiko Shimizu, Yumiko Teramoto, andHayumi Tsunoda for their technical assistance.

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., Iida, A., Iida, S., T., Nishikawa, S., Nagai, R.,
and Nabeshima, Y. (1997) Nature 390, 45–51.
2. Grabnitz, F., Seiss, M., Rucknagel, K. P., and Staudenbauer,
W. L. (1991) Eur. J. Biochem. 200, 301–309.
3. Mantei, N., Villa, M., Enzler, T., Wacker, H., Boll, W., James, P.,
Hunziker, W., and Semenza, G. (1988) EMBO J. 7, 2705–2713.
4. Matsumura, Y., Aizawa, H., Iida, S., T., Nagai, R., Kuro-o, M., andNabeshima, Y. (1998) Biochem. Biophys. Res. Commun. 242, 626 –630.
5. Iida, S., T., Aizawa, H., Matsumura, Y., Sekine, S., Iida, A.,
Anazawa, H., Nagai, R., Kuro-o, M., and Nabeshima, Y. (1998)
FEBS Lett. 424, 6 –10.
6. Aizawa, H., Saito, Y., Nakamura, T., Inoue, M., Imanari, T.,
Ohyama, Y., Matsumura, Y., Masuda, H., Oba, S., Mise, N.,
Kimura, K., Hasegawa, A., Kurabayashi, M., Kuro-o, M., Nabeshima, Y., and Nagai, R. (1998) Biochem. Biophys. Res. Commun. 249, 865– 871.
7. Nishi, T., and Itoh, S. (1986) Gene 44, 29 –36.
8. Hanai, N., Shitara, K., and Yoshida, H. (1986) Cancer Res. 46,
5206 –5210.
9. Lee, K., Deeds, J. D., and Segre, G. V. (1995) Endocrinology 136,
453– 463.

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