Abstract：
Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the CTNS gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate MFSD12 mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of MFSD12 knockout on cystine levels. We showed similar MFSD12 mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC (CTNSΔ57kb) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of mfsd12a translation-blocking morpholino (TB MO) in a ctns-/- zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the mfsd12a TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, MFSD12 mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the ctns-/- zebrafish embryo. In addition, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development warrants further evaluation.NEW & NOTEWORTHY In this study, we show that MFSD12 depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and ctns-/- zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the ctns-/- zebrafish embryos injected with mfsd12a translation-blocking morpholino. Furthermore, a negative effect of the mfsd12a morpholino on the zebrafish development warrants further investigation.
摘要：
胱抑素病是一种常染色体隐性的溶酶体贮积症，由CTNS基因突变引起的，导致cystinosin（CTNS）蛋白缺失或改变。胱抑素从溶酶体中输出胱氨酸,导致胱氨酸积累的功能障碍和其他胱抑素介导的途径的缺陷。胱抑素病是一种全身性疾病,但肾脏是受影响最严重的第一个器官.在肾脏，该疾病最初表现为近端小管的全身性功能障碍（也称为肾Fanconi综合征）。MFSD12是一种溶酶体半胱氨酸导入蛋白，直接影响黑素瘤细胞中的胱氨酸水平，HEK293T细胞,和胱氨酸病患者来源的成纤维细胞。在这项研究中,我们的目的是评估胱氨酸病患者来源的近端肾小管上皮细胞(ciPTEC)中MFSD12mRNA水平,并研究MFSD12基因敲除对胱氨酸水平的影响.与对照细胞相比，我们在患者来源的ciPTEC中显示出相似的MFSD12mRNA表达。患者来源的ciPTEC(CTNSΔ57kb)中的CRISPRMFSD12敲除导致胱氨酸水平显著降低。此外，在ctn-/-斑马鱼模型中,我们评估了注射mfsd12a翻译阻断吗啉代(TBMO)后的近端肾小管重吸收.这导致胱氨酸水平降低，但导致胚胎畸形的浓度依赖性增加。此外，mfsd12aTBMO注射液不能改善近端肾小管重吸收或megalin表达.总之,MFSD12mRNA的消耗降低了两种测试模型中的胱氨酸水平，而没有改善ctn-/-斑马鱼胚胎的近端肾小管功能。此外,较高的mfsd12aTBMO浓度对斑马鱼发育的明显毒性值得进一步评估。在这项研究中，我们表明，MFSD12耗竭与CRISPR/Cas9介导的基因编辑或翻译阻断吗啉显著降低胱氨酸病ciPTEC和ctns-/-斑马鱼胚胎中的胱氨酸水平，分别。然而,我们在注射mfsd12a翻译阻断吗啉代的ctns-/-斑马鱼胚胎中观察到右旋糖酐的近端肾小管重吸收没有改善。此外，mfsd12a吗啉对斑马鱼发育的负面影响值得进一步研究。
