Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany.
Department of Ear, Nose, and Throat Diseases, University Medicine Greifswald, Greifswald, Germany.
FASEB J. 2019 Dec;33(12):14450-14460. doi: 10.1096/fj.201900978RR. Epub 2019 Nov 1.
Hypertension is one of the central causes of kidney damage. In the past it was shown that glomerular hypertension leads to morphologic changes of podocytes and effacement and is responsible for detachment of these postmitotic cells. Because we have shown that podocytes are mechanosensitive and respond to mechanical stress by reorganization of the actin cytoskeleton , we look for mechanotransducers in podocytes. In this study, we demonstrate that the extracellular matrix protein fibronectin (Fn1) might be a potential candidate. The present study shows that Fn1 is essential for the attachment of podocytes during mechanical stress. By real-time quantitative PCR as well as by liquid chromatography-mass spectrometry, we found a significant up-regulation of Fn1 caused by mechanical stretch (3 d, 0.5 Hz, and 5% extension). To study the role of Fn1 in cultured podocytes under mechanical stress, Fn1 was knocked down (Fn1 KD) by a specific small interfering RNA. Additionally, we established a Fn1 knockout (KO) podocyte cell line (Fn1 KO) by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). During mechanical stress, a significant loss of podocytes (>80%) was observed in Fn1 KD as well as Fn1 KO podocytes compared with control cells. Furthermore, Fn1 KO podocytes showed a significant down-regulation of the focal adhesion proteins talin, vinculin, and paxillin and a reduced cell spreading, indicating an important role of Fn1 in adhesion. Analyses of kidney sections from patients with diabetic nephropathy have shown a significant up-regulation of FN1 in contrast to control biopsies. In summary, we show that Fn1 plays an important role in the adaptation of podocytes to mechanical stress.-Kliewe, F., Kaling, S., Lötzsch, H., Artelt, N., Schindler, M., Rogge, H., Schröder, S., Scharf, C., Amann, K., Daniel, C., Lindenmeyer, M. T., Cohen, C. D., Endlich, K., Endlich, N. Fibronectin is up-regulated in podocytes by mechanical stress.
高血压是导致肾脏损伤的主要原因之一。过去的研究表明，肾小球高血压会导致足细胞形态发生变化和足突融合，并导致这些有丝分裂后细胞的脱落。由于我们已经表明足细胞是机械敏感的，并通过肌动蛋白细胞骨架的重组对机械应激做出反应，因此我们在足细胞中寻找机械转导蛋白。在这项研究中，我们证明细胞外基质蛋白纤连蛋白（Fn1）可能是一个潜在的候选者。本研究表明，Fn1 是足细胞在机械应激下附着所必需的。通过实时定量 PCR 以及液相色谱-质谱联用技术，我们发现机械拉伸（3 d、0.5 Hz 和 5%延伸）会显著上调 Fn1。为了研究 Fn1 在机械应激下培养的足细胞中的作用，我们通过特定的小干扰 RNA 敲低 Fn1（Fn1 KD）。此外，我们通过成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关蛋白 9（Cas9）建立了 Fn1 敲除（Fn1 KO）足细胞系（Fn1 KO）。在机械应激下，与对照细胞相比，Fn1 KD 以及 Fn1 KO 足细胞的足细胞丢失率显著升高（>80%）。此外，Fn1 KO 足细胞的黏着斑蛋白塔林、 vinculin 和 paxillin 的表达显著下调，细胞铺展面积减小，表明 Fn1 在黏附中起着重要作用。对糖尿病肾病患者肾组织切片的分析表明，FN1 的表达在糖尿病肾病患者中明显上调，与对照活检相比。总之，我们表明 Fn1 在足细胞适应机械应激中起重要作用。
