The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell-matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell-cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell-cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.

中文翻译：

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肾近端小管模型，用于评估基底膜硬化对肾小管上皮细胞的影响。

肾小管由单层上皮细胞组成，由管状基底膜 (TBM) 支撑，这是一层薄薄的特殊细胞外基质 (ECM)。ECM 的机械特性对于调节广泛的细胞功能很重要，包括增殖、分化和细胞存活。ECM 刚度增加在促进多种病理状况（包括癌症、纤维化和心脏病）中发挥作用。TBM 力学的变化如何调节管状上皮细胞行为尚不完全清楚。在这里，我们介绍了一种细胞培养系统，该系统利用体内衍生的 TBM 来研究肾近端小管细胞中的细胞-基质相互作用。使用生物相容性交联剂京尼平控制基底膜力学。京尼平修饰导致基质硬度呈剂量依赖性增加。交联对 TBM 的扩散分子传输特性具有边际但统计上显着的影响，这可能是由于孔径的减小。天然和京尼平修饰的 TBM 底物都支持肾小管上皮细胞的生长。细胞能够附着并增殖形成融合的单层细胞。管状上皮细胞极化并组装有组织的细胞-细胞连接。京尼平修饰对细胞活力和增殖的影响极小。京尼平硬化 TBM 增加了促纤维化细胞因子的基因表达，并改变了 N-钙粘蛋白的基因表达，N-钙粘蛋白是一种近端肾小管上皮特异性细胞-细胞连接标记物。这项工作介绍了一种新的细胞培养模型，用于利用体内衍生的基底膜进行细胞基底膜力学生物学研究。我们还证明 TBM 硬化通过改变细胞特异性分化标志物的基因表达和诱导促纤维化生长因子的表达增加来影响肾小管上皮细胞的功能。