物理環境對於細胞的生理功能有很重要的影響。之前的研究指出，膠原蛋白的軟度會引起focal adhesion complex 蛋白的降低調控。軟基材也會擾亂腎臟上皮細胞鈣離子的平衡且活化-calpain。 而鈣離子的變化也可以經由細胞鞭毛因流力而彎曲所形成。另外，focal adhesion kinase (FAK) 在胎鼠腎臟發育晚期中漸漸被分解，且在老鼠出生後快速的消失。這其中的機制是如何調控的至今仍未有答案。 我們提出了兩個可能由機械力在腎臟發育過程對於focal adhesion complex 蛋白降低調控的假設：經由降低腎臟本身的軟度或因尿液流力而彎曲鞭毛的影響。第一步我們先利用動態機械分析儀和原子力顯微鏡來測量腎臟在不同發育階段的軟硬度。結果顯示腎臟的軟硬度在出生後是上升的。因此我們提出FAK在腎臟發育過程的降解是因為鞭毛被尿液流力彎曲，因而使得細胞內鈣離子的上升，活化-calpain 並切FAK。免疫組織染色顯示只有在胎鼠19.5日後才能從腎小管中找到鞭毛，而這個時間點和FAK 在腎臟發育過程開始被分解的時間是相同的。為了要模擬腎臟內尿液的流動，我們給予MDCK分支3B5細胞 4 dyne/cm2的剪流力。我們發現在30分鐘內focal adhesion complex 蛋白會被分解，而 -spectrin也會產生145 kD和120 kD的片段。這些結果的模式和發育中的腎臟相似，反映活化calpain和caspase-3的可能性。另一方面，將細胞擺在每秒20次的旋轉機或擺動機上所造成的雙方向或旋轉性流力都能使得FAK隨時間降低調控。螢光免疫染色顯示當剪流力增強時 (2, 4, 6 dyne/cm2)，FAK會從細胞周圍轉移到細胞內。 這個研究提供機械力如何在腎臟發育過程中調控focal adhesion complex 蛋白的可能性。

Physical environment plays an important role in modulating cellular physiological functions. Previous study showed that low rigidity of collagen gel induced downregulation of focal adhesion complex proteins. Soft substrate can also disturb calcium homeostasis and activate -calpain in renal epithelial cells. It has been shown that intracellular calcium concentration is altered when primary cilia on kidney epithelial cells sense shear flow. The bending of primary cilia increases intracellular calcium concentration. Our previous studies have found that focal adhesion kinase (FAK) was degraded gradually in late embryonic rat kidney development and rapidly diminished after birth, yet the regulating mechanism was unclear. We hypothesized two possibilities of how mechanical force regulates focal adhesion complex proteins in developing kidney: via decreasing kidney rigidity after birth or sensing urine flow by the primary cilia. We first measured the elasticity of kidney in different developing stages using dynamic mechanical analyzer and atomic force microscopy. The results showed that the rigidity was increased after birth. We therefore proposed that degradation of FAK in the developing kidney might be caused by urine shear flow mediated by primary cilium of epithelium. Immunohistochemical study showed that cilia formation was found in kidney tubules as early as at embryonic day 19.5 during development, which matched our initial observation in FAK degradation in embryonic kidney. In order to mimic urine flow in the kidney, we applied luminal shear flow (4 dyne/cm2) to MDCK subline 3B5 cells. We observed degradation of focal adhesion complex proteins and degraded fragments of -spectrin at 145 and 120 kD within 30 min. These results were similar as in the developing kidney, suggesting possible activation of calpain and caspase-3. On the other hand, bidirectional and rotational stress, produced by placing cultured cells on a shaker or rotator at 20 rpm, respectively, resulted in downregulation of FAK in time dependent manner. Immunofluorescence studies showed that as flow shear stress increased (2, 4, 6 dyne/cm2), FAK translocated from cell periphery to cytosol. This study provided a possible mechanism on how mechanical force regulates focal adhesion complex proteins during kidney development.

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