Louzao-Martinez Laura, van Dijk Christian G M, Xu Yan Juan, Korn Amber, Bekker Nicolaas J, Brouwhuis Romi, Nicese Maria Novella, Demmers Jeroen A A, Goumans Marie-José T H, Masereeuw Rosalinde, Duncker Dirk J, Verhaar Marianne C, Cheng Caroline
Department of Nephrology and Hypertension, University Medical Center Utrecht, the Netherlands.
Proteomics Center, Erasmus University Medical Center, the Netherlands.
Matrix Biol Plus. 2019 Jul 25;4:100011. doi: 10.1016/j.mbplus.2019.100011. eCollection 2019 Nov.
Cell-based approaches using tissue engineering and regenerative medicine to replace damaged renal tissue with 3D constructs is a promising emerging therapy for kidney disease. Besides living cells, a template provided by a scaffold based on biomaterials and bioactive factors is needed for successful kidney engineering. Nature's own template for a scaffolding system is the extracellular matrix (ECM). Research has focused on mapping the mature renal ECM; however, the developing fetal ECM matches more the active environment required in 3D renal constructs. Here, we characterized the differences between the human fetal and mature renal ECM using spectrometry-based proteomics of decellularized tissue. We identified 99 different renal ECM proteins of which the majority forms an overlapping core, but also includes proteins enriched in either the fetal or mature ECM. Relative protein quantification showed a significant dominance of EMILIN1 in the fetal ECM. We functionally tested the role of EMILIN1 in the ECM using a novel methodology that permits the reliable anchorage of native cell-secreted ECM to glass coverslips. Depletion of EMILIN1 from the ECM layer using siRNA mediated knock-down technologies does not affect renal epithelial cell growth, but does promote migration. Lack of EMILIN1 in the ECM layer reduces the adhesion strength of renal epithelial cells, shown by a decrease in focal adhesion points and associated stress fibers. We showed in this study the importance of a human renal fetal and mature ECM catalogue for identifying promising ECM components that have high implementation potential in scaffolds for 3D renal constructs.
利用组织工程和再生医学，通过3D构建体替代受损肾组织的基于细胞的方法，是一种很有前景的新兴肾脏疾病治疗方法。除了活细胞外，成功进行肾脏工程还需要基于生物材料和生物活性因子的支架提供的模板。自然界自身的支架系统模板是细胞外基质（ECM）。研究主要集中在绘制成熟肾ECM图谱；然而，发育中的胎儿ECM更符合3D肾构建体所需的活跃环境。在这里，我们使用基于光谱的脱细胞组织蛋白质组学，表征了人胎儿和成熟肾ECM之间的差异。我们鉴定出99种不同的肾ECM蛋白，其中大多数形成一个重叠的核心，但也包括在胎儿或成熟ECM中富集的蛋白质。相对蛋白质定量显示，EMILIN1在胎儿ECM中占显著优势。我们使用一种新方法对EMILIN1在ECM中的作用进行了功能测试，该方法允许将天然细胞分泌的ECM可靠地锚定在玻璃盖玻片上。使用siRNA介导的敲低技术从ECM层中去除EMILIN1，并不影响肾上皮细胞的生长，但确实促进了其迁移。ECM层中缺乏EMILIN1会降低肾上皮细胞的黏附强度，表现为黏着斑和相关应力纤维数量减少。我们在本研究中表明，建立人胎儿和成熟肾ECM目录对于识别在3D肾构建体支架中具有高应用潜力的有前景的ECM成分非常重要。
