Enzyme Replacement Therapy Clears Gb3 Deposits from a Podocyte Cell Culture Model of Fabry Disease but Fails to Restore Altered Cellular Signaling.
Enzyme Replacement Therapy Clears Gb3 Deposits from a Podocyte Cell Culture Model of Fabry Disease but Fails to Restore Altered Cellular Signaling.
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DOI：
10.33594/000000077
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Kurschat, Christine E
中科院分区：
文献类型：
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作者：
Braun, Fabian;Blomberg, Linda;Kurschat, Christine E
背景/目的：法布里病 (FD) 是一种溶酶体贮积症，其特征是由于 GLA 基因突变导致 α-半乳糖苷酶 A (α-Gal A) 酶活性受损。虽然几乎所有组织都受到影响，但肾脏损伤对于患者的预后尤其重要。目前，缺乏强大的诊断工具和研究肾脏FD的体内研究模型，这是进一步改进诊断和治疗的主要障碍。本研究的重点是酶替代疗法对先前建立的法布里病足细胞细胞培养模型的影响。方法：我们模拟酶替代疗法，研究了体外应用α-Gal A对法布里足细胞3天的影响。我们通过免疫荧光、电子显微镜、串联质谱和蛋白质印迹研究了 Gb3 水平的降低和失调的分子途径，如自噬、mTOR/AKT 信号传导和促纤维化信号传导。结果：我们检测到 α-Gal A 处理后法布里足细胞中 Gb3 积累的完全消退。尽管 Gb3 清除能力很强，但所研究的信号通路失调并未逆转。结论：本研究首次提供了关于 FD 信号转导机制失调在 α-Gal A 治疗后未恢复的 Gb3 独立效应的证据。我们假设在 FD 中观察到的细胞内改变可能有一个不可逆转的点，此后尽管 Gb3 被清除，但通过 α-Gal A 治疗逆转失调的细胞信号转导是无效的。我们的观察结果表明，需要进一步研究法布里足细胞改变的信号转导机制，并确定开始法布里治疗的适当时间。
BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by impaired alpha-galactosidase A (alpha-Gal A) enzyme activity due to mutations in the GLA gene. While virtually all tissues are affected, renal damage is particularly critical for the patients' outcome. Currently, powerful diagnostic tools and in vivo research models to study FD in the kidney are lacking, which is a major obstacle for further improvements in diagnosis and therapy. The present study focuses on the effects of enzyme replacement therapy on a previously established podocyte cell culture model of Fabry disease.METHODS: We investigated the effect of in vitro application of alpha-Gal A on Fabry podocytes for 3 days, mimicking enzyme replacement therapy. We studied reduction of Gb3 levels and dysregulated molecular pathways such as autophagy, mTOR/AKT signaling and pro-fibrotic signaling by employing immunofluorescence, electron microscopy, tandem mass spectrometry and western blot.RESULTS: We detected complete resolution of Gb3 accumulation in Fabry podocytes upon alpha-Gal A treatment. Despite robust Gb3 clearance, dysregulation of the signaling pathways investigated was not reversed.CONCLUSION: This study presents first evidence for Gb3-independent effects regarding dysregulation of signal transduction mechanisms in FD not recovering upon alpha-Gal A treatment. We assume that intracellular alterations observed in FD may have a point of no return after which a reversal of dysregulated cellular signal transduction by alpha-Gal A treatment is not effective, despite Gb3 clearance. Our observations suggest further research on signal transduction mechanisms altered in Fabry podocytes and on determining the appropriate time for initiation of Fabry therapy.