Yamamoto H, Tsuruoka S, Ioka T, Ando H, Ito C, Akimoto T, Fujimura A, Asano Y, Kusano E
Department of Nephrology, Jichi Medical School, Tochigi, Japan.
Kidney Int. 2006 May;69(10):1780-5. doi: 10.1038/sj.ki.5000340.
Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of arteriosclerosis. Clinical studies show that uremic toxins deteriorate the arteriosclerosis in renal failure patients. Indoxyl sulfate (IS) is a strong protein-bound uremic toxin, but the effect of IS on VSMC proliferation has not been studied. We examined the effect of IS on rat VSMC proliferation, assessed by a cell counting kit (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] assay) and by [(3)H]thymidine incorporation in vitro. We further evaluated a contribution of mitogen-activated protein kinase (MAPK; p44/42 MAPK) to VSMC proliferation by IS. Immunohistochemical staining was performed for VSMCs using antirat organic anion transporter (OAT)3 antibody. The mRNA expressions of platelet-derived growth factor (PDGF)-A and -C chains, and PDGF-beta receptor were evaluated by real-time PCR. IS stimulated the proliferation of VSMCs in a concentration-dependent manner and activated p44/42 MAPK. Concentration of IS needed to stimulate the proliferation of rat VSMC was about 250 microM, which is compatible with that in the serum of end-stage renal failure patients. PD98059 (10 microM), a selective inhibitor of MAPK/extracellular signal-regulated kinase, inhibited the IS-induced (250 microM) VSMC proliferation and phosphorylation of MAPK. Probenecid (0.5 mM), an inhibitor and substrate of OAT, inhibited the IS-induced (250 microM) VSMC proliferation. Rat OAT3 was detected in VSMCs. The mRNA expressions of PDGF-C chain and PDGF-beta receptor were significantly increased by IS. We conclude that IS directly stimulates rat VSMC proliferation and activates MAPK in vitro. This might be one of the mechanisms underlying the progression of atherosclerotic lesions in end-stage renal disease patients.
血管平滑肌细胞（VSMC）增殖是动脉粥样硬化进展中的关键事件。临床研究表明，尿毒症毒素会使肾衰竭患者的动脉粥样硬化恶化。硫酸吲哚酚（IS）是一种与蛋白质紧密结合的强尿毒症毒素，但IS对VSMC增殖的影响尚未得到研究。我们通过细胞计数试剂盒（4-[3-[4-碘苯基]-2-4(4-硝基苯基)-2H-5-四氮唑]-1,3-苯二磺酸盐]测定法）和体外[³H]胸腺嘧啶核苷掺入法，检测了IS对大鼠VSMC增殖的影响。我们还进一步评估了丝裂原活化蛋白激酶（MAPK；p44/42 MAPK）对IS诱导的VSMC增殖的作用。使用抗大鼠有机阴离子转运体（OAT）3抗体对VSMC进行免疫组织化学染色。通过实时PCR评估血小板衍生生长因子（PDGF）-A和-C链以及PDGF-β受体的mRNA表达。IS以浓度依赖性方式刺激VSMC增殖并激活p44/42 MAPK。刺激大鼠VSMC增殖所需的IS浓度约为250微摩尔，这与终末期肾衰竭患者血清中的浓度相当。MAPK/细胞外信号调节激酶的选择性抑制剂PD98059（10微摩尔）可抑制IS诱导的（250微摩尔）VSMC增殖和MAPK磷酸化。丙磺舒（0.5毫摩尔）是OAT的抑制剂和底物，可抑制IS诱导的（250微摩尔）VSMC增殖。在VSMC中检测到大鼠OAT3。IS可显著增加PDGF-C链和PDGF-β受体的mRNA表达。我们得出结论，IS在体外直接刺激大鼠VSMC增殖并激活MAPK。这可能是终末期肾病患者动脉粥样硬化病变进展的潜在机制之一。
