Can klotho alleviate muscle fibrosis in muscular dystrophy?

描述（由申请人提供）：杜氏肌营养不良症（DMD）与肌营养不良蛋白基因突变有关，导致横纹肌进行性萎缩和早期死亡。DMD患者的骨骼肌经历肌纤维坏死和再生的循环，但随着时间的推移，肌纤维会丢失，退化的肌纤维被纤维化组织取代。治疗DMD需要稳定肌纤维肌膜和对抗纤维化的手段。虽然这两个方面都得到了深入的研究，但有效的治疗方法还有待开发。拟议的研究将分析klotho（也称为alpha-klotho）减少肌营养不良蛋白缺失mdx小鼠（一种肌营养不良症小鼠模型）纤维化的潜力。Klotho作为跨膜蛋白和分泌的体液因子两者起作用。分泌的klotho已显示直接抑制肾组织中的促纤维化TGF-β 1和Wnt途径。虽然klotho表达的主要部位是肾脏，但它也在其他组织中表达。如果klotho的抗纤维化作用延伸到肌肉组织，则klotho蛋白可以成为减少肌营养不良蛋白缺乏症中的肌肉纤维化的有趣的候选物，其中已经确定了TGF-β 1和Wnt在肌肉纤维化中的作用。该项目的一个主要目的是深入了解klotho转基因（Kt）过表达减轻mdx小鼠肌肉纤维化的潜力。除了纤维化、钙化和肌纤维病理学的组织学测定外，该研究还将检查CD 45+细胞水平，作为炎症和肌原性（卫星细胞）活性的指标，这些活性似乎受到纤维化的损害。第二个目的是通过检查mdx小鼠中下降的自愿轮跑来分析Kt转基因对全身表现的影响。将使用产生比“标准”mdx小鼠品系更少的回复突变肌纤维的mdx 4cv品系。重点将放在年轻至老年的mdx和Kt/mdx小鼠组中，以全面了解mdx肌肉病理学随年龄增长而加剧。已经有了用于拟议研究的鼠标线和专门知识，可以取得有效进展。人们认识到，虽然mdx小鼠已被广泛用作DMD的实验室模型，但mdx病理学不如DMD患者严重。尽管如此，对mdx小鼠的研究为后续的营养不良犬的研究和人类的初步临床试验提供了重要的指导数据。在所提出的研究中另外包括野生型（wt）和Kt/wt小鼠组将不仅允许与亲本系进行比较，而且允许深入了解klotho转基因在野生型背景下的作用。这项探索性研究的结果可能为防治DMD和其他肌营养不良症以及改善衰老中的肌肉质量确定一个新的临床方向。R21机制旨在鼓励探索性高风险高回报的新研究，是这一应用的最佳平台。

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is associated with mutations in the dystrophin gene, leading to progressive wasting of striated muscle and early death. Skeletal muscles in DMD patients undergo cycles of myofiber necrosis and regeneration, but over time there is a loss of myofibers and the degenerating myofibers are replaced with fibrotic tissue. Treating DMD requires means for stabilizing the myofiber sarcolemma and for combating fibrosis. Although both fronts have been intensely studied, effective treatments are yet to be developed. The proposed study will analyze the potential of klotho (also known as alpha-klotho) to reduce fibrosis in the dystrophin-null mdx mouse, a murine model of muscular dystrophy. Klotho functions as both a transmembrane protein and a secreted humoral factor. The secreted klotho has been shown to directly inhibit the pro-fibrotic TGF-ß1 and Wnt pathways in renal tissue. While the main site of klotho expression is the kidney, it is also expressed in other tissues. Should the anti-fibrotic effect of klotho extend to he muscle tissues, the klotho protein could become an intriguing candidate for reducing muscle fibrosis in dystrophin-deficiency where a role for TGF-ß1 and Wnt in muscle fibrosis has been established. One main aim of the proposed project is to gain insight into the potential of klotho transgenic (Kt) overexpression to alleviate muscle fibrosis in mdx mice. In addition to histological assays of fibrosis, calcification and myofiber pathology, the study will examine CD45+ cell level as a measure of inflammation and myogenic (satellite cell) activity that appears to be impaired by fibrosis. A second aim is to analyze the effect of the Kt transgene on whole body performance by examining voluntary wheel running, which declines in the mdx mouse. The mdx4cv strain that develops less revertant myofibers than the "standard" mdx mouse line will be used. Emphasis will be given to mdx and Kt/mdx mouse groups of young to old ages to gain a comprehensive insight as the mdx muscle pathology intensifies with age. Both the mouse lines and the expertise for the proposed studies are already available, permitting effective progress. It is recognized that while the mdx mouse has been used extensively as a laboratory model of DMD, the mdx pathology is less severe than that of DMD patients. Still, studies with mdx mice have generated important directive data for subsequent studies with dystrophic dogs and for pilot clinical trials in humans. The additional inclusion of wildtype (wt) and Kt/wt mouse groups in the proposed study will not only permit comparison with the parental lines, but also allow gaining insights into the effect of the klotho transgene in the wildtype context. The outcome of this exploratory study may identify a new clinical direction for combating DMD and other muscular dystrophies as well as improving muscle quality in aging. The R21 mechanism that is intended to encourage exploratory high-risk high-reward novel studies is the optimal platform for this application.