Supplementary Table1 (Urakawa I. et al)

2005-05-05504E

Supplementary Table 1. FGF23 response with or without Klotho transfection in various cell lines

Cell line (origin) / FGF23 responsea, b (without Klotho) / FGF23 responseb (with Klotho transfection) / Detection of Klotho protein after transfectionc
HEK293 (human kidney) / - / + / +
HKC-8 (human kidney) / - / + / +
CHO (hamster ovary) / - / + / +
ATDC5 (mouse chondrocyte) / - / + / N.D.
MC3T3-E1 (mouse osteoblast) / - / + / N.D.
L6 (rat myocyte) / - / - # / +
BaF3 (mouse pro-B cell) / - / - / -
AtT-20/D16c-F2 (mouse pituitary) / - / - / -
MDCK (canine kidney) / - / - / -
NRK49F (rat kidney) / - / - / -
S13 (mouse kidney) / - / - / -

a. ERK1/2 activation was examined 10 min after treatment with 100 ng/mL FGF23 (n = 3). b. Cells were transfected with the Egr-1-luciferase reporter plasmid and the Klotho expression plasmid. The positive response was determined by a significant increase in luciferase activity by treatment with 100 ng/mL FGF23 (n = 3). c. Cells were transfected with the Klotho expression plasmid and cultured for 48 h, then subjected to Western blot analysis with anti-Klotho polyclonal antibodies. #, FGF23 response is reproducible when Klotho and FGFR1(IIIc) were co-expressed. N.D., not determined.

Supplementary Table 2. Serum parameters of FGF23-null mice, Klotho-deficient mice, and anti-Klotho monoclonal antibody-treated mice.

+/+ / Fgf23(-/-) / +/+ / kl/kl / Anti-Klotho Antibody Treatement
9 h 24 h
Control / -Klotho Ab / Control / -Klotho Ab
1,25(OH)2D (pg/mL) / 115 ± 11 / 349 ± 39** / 129 ± 8 / 384 ± 60** / 129 ± 19 / 462 ± 36** / 113 ± 18 / 138 ± 9
Phosphate (mg/dL) / 9.30 ± 0.53 / 14.47 ± 0.54** / 9.00 ± 0.26 / 13.90 ± 0.23** / 7.92± 0.53 / 7.22 ± 0.14 / 7.60 ± 0.47 / 9.08 ± 0.35**
Calcium (mg/dL) / 8.88 ± 0.27 / 9.87 ± 0.30* / 9.03 ± 0.11 / 10.17 ± 0.25** / 9.18 ± 0.11 / 9.26 ± 0.13 / 9.42 ± 0.05 / 9.80 ± 0.10**
PTH (pg/mL) / 13.7 ± 3.5 / 0.8 ± 0.5** / 19.3 ± 4.8 / 7.5 ± 5.0 / 29.8 ± 15.3 / 15.5 ± 5.0 / 24.3 ± 12.1 / 12.1 ± 5.1

Serum samples were prepared from 6 week-old Fgf23(-/-), Klotho-deficient (kl/kl), and respective control (+/+) littermates (n=6 each). Samples were also collected from 8 week-old wild-type mice treated with vehicle (control) or anti-Klotho antibody at 9 and 24 h post injection (n=5 each). *P<0.05, **P<0.01 compared to each control by Student’s t-test. Data are expressed as mean ± SEM.

Supplementary Table 3. Serum parameters including FGF23 and renal expressions of key enzymes for vitamin D metabolism in Klotho-insufficient (kl/+) and Klotho-deficient (kl/kl) mice.

Serum parameters / WT
(n=8) / kl/+
(n=7) / kl/kl
(n=4)
1,25(OH)2D (pg/mL) / 85 ± 6 / 91 ± 4 / 249 ± 23**
Phosphate (mg/dL) / 8.9 ± 0.3 / 10.1 ± 0.5 / 14.4 ± 0.3**
Calcium (mg/dL) / 9.3 ± 0.2 / 9.4 ± 0.1 / 11.6 ± 0.1**
FGF23 (pg/mL) / 151 ± 16 / 252 ± 28** / 249000 ± 61000**
mRNA expressions / WT
(n=6) / kl/+
(n=6) / kl/kl
(n=6)
Cyp27b1 / -actin / 1.00 ± 0.23 / 0.76 ± 0.14 / 2.95 ± 0.68*
Cyp24 / -actin / 1.00 ± 0.24 / 1.51 ± 0.16 / 2.29 ± 0.43*
Klotho / -actin / 1.00 ± 0.04 / 0.57 ± 0.03** / 0.00 ± 0.00**

Six week-old wild-type, Klotho-insufficient (kl/+), and Klotho-deficient (kl/kl) mice were anesthetized to collect serum samples and then sacrificed to prepare renal mRNAs. Relative mRNA abundance of Cyp27b1, Cyp24, and Klotho compared to -actin was determined by real-time quantitative PCR. *P<0.05, **P<0.01 compared to wild-type by Student’s t-test. Data are expressed as mean ± SEM.

Supplementary Table 4. Serum parameters and relative mRNA expressions of littermates obtained from three pairs of Fgf23(+/-) and Klotho-insufficient (kl/+) mice.

WT
(n=9) / Fgf23(+/-)
(n=4) / kl/+
(n=4) / Fgf23(+/-) (kl/+)
(n=9)

Serum parameters

1,25(OH)2D (pg/mL) / 96 ± 33 / 91 ± 30 / 102 ± 31 / 105 ± 30
Phosphate (mg/dL) / 8.1 ± 1.0 / 8.9 ± 0.6 / 8.1 ± 0.9 / 8.9 ± 0.6
Calcium (mg/dL) / 9.6 ± 0.2 / 9.8 ± 0.2 / 9.5 ± 0.2 / 9.5 ± 0.2
FGF23 (pg/mL) / 89 ± 8 / 79 ± 3 / 118 ± 21a / 99 ± 11 b

Relative mRNA expression

Cyp27b1 / -actin / 1.0 ± 0.5 / 0.9 ± 0.2 / 1.5 ± 0.3 / 1.2 ± 0.3
Cyp24 / -actin / 1.0 ± 0.7 / 0.6 ± 0.2 / 1.0 ± 0.6 / 1.0 ± 0.7
Klotho / -actin / 1.0 ± 0.2 / 1.1 ± 0.1 / 0.6 ± 0.2c / 0.6 ± 0.1c

Male Klotho-insufficient (kl/+) mice and female heterozygous Fgf23(+/-) mice were crossed and the genotype of obtained littermates was determined by a combination of genomic PCRs using the primer sets for wild-type Klotho (5’-TTGTGGAGATTGGAAGTGGACGAAAGAG-3’ and 5’-CTGGACCCCCTGA

-AGCTGGAGTTAC-3’), mutant Klotho (5’-TTGTGGAGATTGGAAGTGGACGAAAGAG-3’ and 5’-CGCCCCGACCGGAGCTGAGAGTA-3’), and FGF2317. Serum samples and kidney mRNAs were prepared from 6 week-old wild-type (WT), Fgf23(+/-), kl/+, and Fgf23(+/-)(kl/+) mice obtained from three pairs of Fgf23(+/-) and Klotho-deficient (kl/+) mice. Relative mRNA expression levels of Cyp27b1, Cyp24, and Klotho normalized to -actinwere determined by quantitative real-time RT-PCRs Data are expressed as mean ± SEM. aP<0.05vs. WT, bP<0.05 vs. Fgf23(+/-) and cP<0.05vs. WT by Student’s t-test.