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，退化的肌纤维会被纤维化组织所取代。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，但有效的治疗方法尚未开发出来。拟议的研究将分析klotho（也称为α -klotho）在肌肉萎缩症小鼠模型mdx中减少抗营养不良蛋白缺失的纤维化的潜力。Klotho既是一种跨膜蛋白，也是一种分泌性体液因子。所分泌的klotho已被证明直接抑制肾组织中促纤维化TGF-ß1和Wnt通路。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的抗纤维化作用扩展到肌肉组织，klotho蛋白可能成为减少肌营养不良蛋白缺乏症中肌肉纤维化的有趣候选蛋白，TGF-ß1和Wnt在肌肉纤维化中的作用已被确立。该项目的一个主要目的是深入了解klotho转基因（Kt）过表达的潜力，以减轻mdx小鼠的肌肉纤维化。除了纤维化、钙化和肌纤维病理的组织学分析外，该研究还将检测CD45+细胞水平，作为炎症和肌原性（卫星细胞）活性的测量指标，这些活性似乎被纤维化损害。第二个目的是分析Kt转基因对全身性能的影响，通过检测mdx小鼠的自主转轮跑能力下降。将使用mdx4cv菌株，它比“标准”mdx小鼠株产生更少的肌纤维。重点是mdx和Kt/mdx小鼠幼龄至老年组，以全面了解mdx肌肉病理随着年龄的增长而加剧。小鼠系和拟议研究的专业知识都已经可用，允许有效的进展。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的病理情况不如DMD患者严重。尽管如此，对mdx小鼠的研究为随后对营养不良犬的研究和对人类的初步临床试验提供了重要的指导数据。在拟议的研究中额外包括野生型（wt）和Kt/wt小鼠组，不仅可以与亲本系进行比较，还可以深入了解klotho转基因在野生型背景下的影响。本探索性研究的结果可能为对抗DMD和其他肌肉营养不良症以及改善衰老过程中的肌肉质量确定新的临床方向。R21机制旨在鼓励探索性高风险高回报的新颖研究，是该应用的最佳平台。

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.