Background: The prevalence of chronic kidney disease (CKD) is increasing worldwide. Patients with CKD and particularly end-stage renal disease (ESRD) are at increased risk of mortality, mainly from cardiovascular disease (CVD).

Vascular calcification (VC) is the depositions of calcium (Ca) phosphate (P) salt in the vasculature. VC in the medial layer, also known as Monckeberg's calcification, is common in CKD patients and is associated with increased risk of CVD. The mechanism of VC is thought to be a highly complex process that shares many similarities with the mineralization of bone. In vivo and in vitro studies have shown that P induces osteoblastic differentiation of vascular smooth muscle cells (VSMCs).

Fibroblast growth factor 23 (FGF23) is the most recently discovered FGF which belongs to the FGF19 subfamily. It is synthesized in bone and acts mainly on the kidney and parathyroid as a P-regulating hormone. The presence of the co-receptor Klotho, which binds FGF receptors (FGFRs), is necessary for induction of the FGF23-specific signaling.

Circulating levels of FGF23 increase as kidney function declines. Clinical studies suggest that increased level of FGF23 is associated with mortality and VC among CKD patients but its direct effect on VSMC is unknown.

Methods: VSMCs were isolated from thoracic aorta of 8-wk-old male Sprague-Dawley rats. The mRNA expressions of FGFRs and Klotho in VSMCs were examined by reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of Klotho in VSMCs and the aorta were analyzed by western blot and immunohistochemistry. Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in normal VSMCs or Klotho-overexpressed VSMCs (Klotho-VSMCs) by recombinant FGF23 (2-10 ng/ml) was analyzed by western blot.

Calcification of Klotho-VSMCs was induced by 5 mM P medium (high-P medium) in the presence or absence of FGF23 (2-10 ng/ml). The Ca depositions were visualized by Alizarin Red S calcification staining solution and quantified by methylxylenol blue method. The mRNA expressions of osteoblastic transcription factors (Msx2 and osterix) and sodium dependent P transporters (Pit 1 and Pit2) were evaluated by real-time RT-PCR.

Results: FGFR 1, 2, 3 and Klotho mRNA were expressed in cultured VSMCs. Klotho protein was detected in the medial layer of the aorta but not in the isolated VSMCs.

ERK 1/2 phosphorylation by FGF23 was increased in Klotho-VSMCs in a dose dependent manner but not in the normal VSMCs. ERK 1/2 phosphorylation by FGF23 in Klotho-VSMCs was inhibited by FGFR1 inhibitor (SU5402) (2-10 μM) or mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (2-10 μM).

FGF23 accelerated P-induced calcification of Klotho-VSMCs in a dose dependent manner. P induced Msx2 and osterix expressions were enhanced by FGF23 and U0126 inhibited the additive effect of FGF23 on the expression of these osteoblastic markers. The expressions of Pit1 and Pit 2 were not affected by either increased P level or FGF23.

Conclusion: We have shown that FGF23 enhances P-induced calcification in Klotho-VSMCs by promoting osteoblastic differentiation and its effect might be mediated by the ERK 1/2 pathway. Our study indicates that we have to take into consideration the effect of FGF23 on VC during CKD patient's management.

本研究は 慢性腎臓病患者の心血管リスク増加の原因として重要な血管石灰化とリン代謝を調節する新規のホルモン様分子であるFGF23の関連を明らかにするため、ラット/マウス大動脈およびラット初代培養血管平滑筋細胞を用いて、FGF23の血管平滑筋石灰化への関与およびその機序の解析を試みたものであり、下記の結果を得ている。

1.ラット初代培養平滑筋細胞(以下血管平滑筋細胞)でのmRNA発現をRT-PCR法により解析した結果、FGF23のシグナル伝達に必要なFGF受容体1,2,3およびKlothoの発現を認めることが示された。

2.ラット大動脈および血管平滑筋細胞のKlotho蛋白発現をウェスタンブロット法により解析した結果、ラット大動脈ではKlotho発現を認めたが、血管平滑筋細胞では発現を認めないことが示された。

3.マウス大動脈の免疫染色の結果、Klotho蛋白は中膜平滑筋層に高発現していることが示された。

4.Klothoを過剰発現した血管平滑筋細胞ではFGF23の既知の下流シグナルであるERKのリン酸化の亢進が認められ、その作用はFGFR1阻害薬およびMEK阻害薬で抑制された。

5.高リン培地により平滑筋細胞の骨細胞様変化、すなわち石灰化が認められた。さらに石灰化染色およびカルシウム沈着の定量により、FGF23はリンの存在下に血管平滑筋細胞石灰化を亢進することが示された。

6.リンおよびFGF23によるERKリン酸化の検討や、MEK阻害薬を用いた骨転写因子mRNA発現の検討からFGF23はERKリン酸化を亢進させることで、骨転写因子の発現を増加させることが示された。

以上、本論文はFGF23受容体結合タンパクであるKlotho蛋白の血管での発現を確認し、さらにFGF23がKlotho, FGF受容体複合体を介して血管に作用すること、リンによる血管平滑筋石灰化を促進すること、その機序にERK 1/2リン酸化亢進を介した骨転写因子発現増加が関与することを明らかにした。

本研究はこれまで未知であったFGF23の血管作用を示しており、今後慢性腎臓病患者の血管石灰化を予防する新規治療法開発につながると考えられ、学位の授与に値するものと考えられる。