Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand
Department of Developmental Biology, University of Pittsburgh, Pennsylvania.
J Am Soc Nephrol. 2020 May;31(5):962-982. doi: 10.1681/ASN.2019070712. Epub 2020 Mar 20.
Mutations in -a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments.
To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls.
Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities.
These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.
编码胱氨酸转运蛋白胱氨酸酶的基因突变导致罕见的常染色体隐性溶酶体贮积病胱氨酸症。研究还表明胱氨酸参与调节 mTORC1 通路，该通路是细胞代谢、增殖、存活和自噬的核心调节剂。在最严重的形式中，胱氨酸症的特征是胱氨酸积累、肾近端小管功能障碍和肾衰竭。由于使用胱氨酸耗竭药物半胱氨酸只能减缓疾病进展，因此迫切需要更好的治疗方法。
为了解决缺乏研究胱氨酸症的良好基于人类的细胞培养模型的问题，我们生成了第一个人类诱导多能干细胞 (iPSC) 和肾脏类器官模型。我们使用多种技术检查胱氨酸症的特征，包括胱氨酸积累、溶酶体大小、自噬途径和细胞凋亡，并对同基因系进行 RNA 测序，以鉴定与对照相比在胱氨酸症模型中差异表达的基因。
与对照组相比，这些胱氨酸症模型表现出更高的胱氨酸水平、增加的细胞凋亡和受损的基础自噬。半胱氨酸治疗可改善这种表型，除了细胞凋亡和基础自噬的异常。我们发现，使用 mTOR 通路抑制剂依维莫司治疗可减少大溶酶体的数量，减少细胞凋亡并激活自噬，但不能挽救胱氨酸负载的缺陷。然而，用半胱氨酸和依维莫司对胱氨酸症 iPSC 或肾脏类器官进行双重治疗可纠正所有观察到的表型异常。
这些观察结果表明，用胱氨酸耗竭药物（如半胱氨酸）和 mTOR 通路抑制剂（如依维莫司）联合治疗可能改善胱氨酸症的治疗。
