受分泌型klotho 调控的Wnt/β-catenin 信号通路在尿毒症高转化骨病骨髓间充质干细胞功能障碍中的作用

Klotho-deficient mice presented activated Wnt/β-catenin signaling pathway in bone, but its mechanism is unclear, which may be related to the decreased effect of secreted klotho on bone. Klotho expressed in kidney dominantly. Our previous data suggested that rats with uremia and high-turnover osteodystrophy presented significantly decreased serum secreted klotho, increased β-catenin expression in bone, and enhanced capacity of transdifferentiation to osteoblast cell in its bone marrow mesenchymal stem cells (BMMSC). This study is designed to investigate the effects of transfection of secreted-type klotho gene on bone expression of various wnts/fzds/dkks molecules and β-catenin, bone microstructure, osteoblast and osteoclast fuctions in Apo-/- uninephrectomized uremia mice on high phosphorus diet, with or without continuous subcutaneous osmotic pump infusion of dkk-1 to block the signaling pathway. Furthermore, we attempt to observe the effects of klotho on fzds/dkks/β-catenin activity and osteoblast transdifferentiation in BMMSC in high phosphorus and PTH environment, with or without selective suppression of Wnt moleculars. The results of this study would provide new ideas to early intervention with renal high-turnover osteodystrophy.

Klotho 缺陷小鼠骨组织Wnt／β-catenin 信号通路明显激活，这与分泌型klotho 骨作用的丧失有关，但其靶细胞及信号调节机制不明。肾是产生klotho 的主要器官。我们发现尿毒症高转化骨病大鼠血清分泌型klotho 下降与骨β-catenin表达增加、骨髓间充质干细胞（BMMSC）向成骨细胞转分化能力的增强明显相关。本研究拟制作ApoE-/-小鼠单肾切除并残肾缺血再灌注尿毒症小鼠模型，使用分泌型klotho 表达载体转染小鼠，检测各种Wnt、fzd、dkk分子与β-catenin表达变化，观察骨结构、BMMSC体外分化能力改变，及皮下渗透泵输注dkk-1对klotho作用的影响。分析klotho 对高磷、高PTH条件培养的BMMSC中Wnt/β-catenin活性及转分化的影响；选择性敲除不同Wnt分子，寻找klotho相关Wnt靶位点。本研究可为干预肾性骨病提供新的思路。

本研究使用分泌型klotho 表达载体转染尿毒症小鼠，通过体内、外研究，观察klotho转染对骨髓间充质干细胞（BMMSC）Wnt/β-catenin活性及其增殖、分化能力的影响。结果发现：（1）pReceive -LV105-分泌型klotho转染后，小鼠肾组织、血清klotho蛋白表达较对照组增加约3~6倍。（2）尿毒症小鼠骨转化及矿化率均有明显增加，klotho转染后二者恢复至正常水平。流式细胞术显示，klotho转染可使尿毒症小鼠BMMSC中高表达的p-β-catenin明显下降，Wnt激动剂SB-216763可部分阻断klotho对BMMSC中p-β-catenin的影响。（3）原代培养发现，尿毒症小鼠BMMSC增殖及分化能力明显增强，Wnt1、Wnt4 mRNA表达显著上调，后者在klotho转染后受到明显抑制。经免疫沉淀法证实，klotho可与Wnt1、Wnt4结合。（4）klotho转染BMMSC后，p-β-catenin表达及β-catenin核转位明显下降，流式细胞术对BMMSC细胞DNA分析发现S期细胞比例明显减少，碱性磷酸酶、骨桥蛋白等成骨细胞分子表达及骨结节形成数目受到显著抑制，提示其增殖、分化能力下降；以上改变可被SB-216763部分逆转。总之，本研究通过体内、外研究发现，分泌型klotho可通过结合Wnt1、Wnt4而抑制Wnt/β- catenin活性，影响尿毒症小鼠BMMS增殖与分化能力，这可能是尿毒症小鼠高转化骨病发生、发展的重要机制。