Braun Fabian, Blomberg Linda, Brodesser Susanne, Liebau Max C, Schermer Bernhard, Benzing Thomas, Kurschat Christine E
Department II of Internal Medicine and Center for Rare Diseases Cologne, University Hospital of Cologne, Cologne, Germany.
Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
Cell Physiol Biochem. 2019;52(5):1139-1150. doi: 10.33594/000000077.
BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by impaired alpha-galactosidase A (α-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.
We investigated the effect of in vitro application of α-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.
We detected complete resolution of Gb3 accumulation in Fabry podocytes upon α-Gal A treatment. Despite robust Gb3 clearance, dysregulation of the signaling pathways investigated was not reversed.
This study presents first evidence for Gb3-independent effects regarding dysregulation of signal transduction mechanisms in FD not recovering upon α-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 α-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.
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。几乎所有组织都会受到影响，但肾脏损害对患者的预后尤为关键。目前，缺乏用于研究肾脏中法布里病的强大诊断工具和体内研究模型，这是诊断和治疗进一步改善的主要障碍。本研究聚焦于酶替代疗法对先前建立的法布里病足细胞培养模型的影响。
我们通过体外应用α-Gal A处理法布里病足细胞3天来模拟酶替代疗法，研究其效果。我们采用免疫荧光、电子显微镜、串联质谱和蛋白质印迹法，研究Gb3水平的降低以及自噬、mTOR/AKT信号传导和促纤维化信号传导等失调的分子途径。
我们检测到α-Gal A处理后法布里病足细胞中Gb3积累完全消退。尽管Gb3得到了有效清除，但所研究的信号通路失调并未得到逆转。
本研究首次证明，法布里病中信号转导机制失调存在与Gb3无关的效应，α-Gal A治疗后无法恢复。我们推测，法布里病中观察到的细胞内改变可能存在一个无法逆转的点，在此之后，尽管Gb3被清除，但α-Gal A治疗对失调的细胞信号转导的逆转无效。我们的观察结果表明，需要进一步研究法布里病足细胞中改变的信号转导机制，并确定法布里病治疗开始的合适时间。
