Barker Sarah L, Pastor Johanne, Carranza Danielle, Quiñones Henry, Griffith Carolyn, Goetz Regina, Mohammadi Moosa, Ye Jianfeng, Zhang Jianning, Hu Ming Chang, Kuro-o Makoto, Moe Orson W, Sidhu Sachdev S
Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada.
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Nephrol Dial Transplant. 2015 Feb;30(2):223-33. doi: 10.1093/ndt/gfu291. Epub 2014 Oct 15.
αKlotho is the prototypic member of the Klotho family and is most highly expressed in the kidney. αKlotho has pleiotropic biologic effects, and in the kidney, its actions include regulation of ion transport, cytoprotection, anti-oxidation and anti-fibrosis. In rodent models of chronic kidney disease (CKD), αKlotho deficiency has been shown to be an early biomarker as well as a pathogenic factor. The database for αKlotho in human CKD remains controversial even after years of study.
We used a synthetic antibody library to identify a high-affinity human antigen-binding fragment that recognizes human, rat and mouse αKlotho primarily in its native, rather than denatured, form.
Using an immunoprecipitation-immunoblot (IP-IB) assay, we measured both serum and urinary levels of full-length soluble αKlotho in humans and established that human CKD is associated with αKlotho deficiency in serum and urine. αKlotho levels were detectably lower in early CKD preceding disturbances in other parameters of mineral metabolism and progressively declined with CKD stages. We also found that exogenously added αKlotho is inherently unstable in the CKD milieu suggesting that decreased production may not be the sole reason for αKlotho deficiency.
Synthetic antibody libraries harbor tremendous potential for a variety of biomedical and clinical applications. Using such a reagent, we furnish data in support of αKlotho deficiency in human CKD, and we set the foundation for the development of diagnostic and therapeutic applications of anti-αKlotho antibodies.
α-klotho是Klotho家族的原型成员，在肾脏中表达最为丰富。α-klotho具有多效性生物学效应，在肾脏中，其作用包括调节离子转运、细胞保护、抗氧化和抗纤维化。在慢性肾脏病（CKD）的啮齿动物模型中，α-klotho缺乏已被证明是一种早期生物标志物以及致病因素。即使经过多年研究，人类CKD中α-klotho的数据库仍存在争议。
我们使用合成抗体库来鉴定一种高亲和力的人源抗原结合片段，该片段主要识别天然形式而非变性形式的人、大鼠和小鼠α-klotho。
使用免疫沉淀-免疫印迹（IP-IB）分析，我们测量了人类血清和尿液中全长可溶性α-klotho的水平，并确定人类CKD与血清和尿液中的α-klotho缺乏有关。在矿物质代谢的其他参数出现紊乱之前的早期CKD中，α-klotho水平明显较低，并随着CKD阶段的进展而逐渐下降。我们还发现，在CKD环境中外源性添加的α-klotho本身不稳定，这表明产量降低可能不是α-klotho缺乏的唯一原因。
合成抗体库在各种生物医学和临床应用中具有巨大潜力。使用这样一种试剂，我们提供了支持人类CKD中α-klotho缺乏的数据，并为抗α-klotho抗体的诊断和治疗应用开发奠定了基础。
