Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis
Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis
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DOI：
10.1681/asn.2019090956
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发表时间：
2020-07-01
影响因子：
13.6
通讯作者：
Rega, Laura Rita
中科院分区：
文献类型：
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作者：
De Leo, Ester;Elmonem, Mohamed A.;Rega, Laura Rita
肾病性胱氨酸病是溶酶体贮积病中最严重的一种，表现为肾范可尼综合征。胱氨酸转运缺陷导致胱氨酸在溶酶体内蓄积和结晶。胱氨酸病的唯一治疗方法半胱胺不能纠正范可尼综合征或防止进展为ESKD，表明涉及与溶酶体胱氨酸蓄积无关的途径。作者使用高通量筛选来鉴定减少自噬底物p62/SQSTM 1在胱氨酸病近端肾小管上皮细胞中积累并恢复正常自噬的分子。在来自胱氨酸病患者或小鼠模型的细胞和胱氨酸病斑马鱼中的研究表明，毛地黄黄酮纠正了与肾范可尼综合征相关的胱氨酸表型的各个方面。这些发现为肾病性胱氨酸病和其他肾脏溶酶体胆积病的治疗提供了新的前景。背景编码溶酶体胱氨酸转运体胱氨酸蛋白的基因突变导致溶酶体胆积病胱氨酸病。胱氨酸转运缺陷导致胱氨酸在溶酶体内蓄积和结晶。最严重的表型，肾病性胱氨酸病，在生命的最初几个月表现为肾范可尼综合征。胱氨酸消耗剂半胱胺可显著延迟症状，但不能阻止ESKD的进展，也不能治疗范可尼综合征。这表明肾病性胱氨酸病中的途径与溶酶体胱氨酸蓄积无关。最近的数据表明，这样一个潜在的途径，溶酶体介导的降解的自噬货物，是妥协的cystinosis.Methods要确定药物，降低水平的自噬相关蛋白p62/SQSTM 1在cystinotic近端肾小管上皮细胞，我们进行了高通量筛选的基础上，在细胞内ELISA检测。然后，我们测试了一个有前途的候选人来自患者的细胞，和小鼠模型，胱氨酸病，并在cystinotic zebrafish.Results的临床前研究确定为减少p62/SQSTM 1水平的胱氨酸细胞的46种化合物，我们选择毛地黄黄酮的基础上，其疗效，安全性和相似性染料木素，我们以前表明，改善其他溶酶体异常的胱氨酸细胞。我们的数据表明毛地黄黄酮改善自噬？溶酶体降解途径，是一种强大的抗氧化剂，并具有抗凋亡特性。此外，毛地黄黄酮刺激内吞作用，提高内吞受体megalin.Conclusions的表达，我们的数据表明，毛地黄黄酮改善有缺陷的途径，胱氨酸病，并具有良好的安全性，因此有可能作为一种治疗肾病性胱氨酸病和其他肾脏溶酶体储存疾病。
Significance Statement Nephropathic cystinosis, the most severe form of the lysosomal storage disease cystinosis, presents as renal Fanconi syndrome. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The only treatment for cystinosis, cysteamine, does not correct the Fanconi syndrome or prevent progression to ESKD, suggesting involvement of pathways unrelated to lysosomal cystine accumulation. The authors used high-throughput screening to identify molecules that reduce accumulation of the autophagy substrate p62/SQSTM1 in cystinotic proximal tubular epithelial cells and restore normal autophagy. Studies in cells derived from patients with, or mouse models of, cystinosis and in cystinotic zebrafish indicate that luteolin corrects aspects of the cystinotic phenotype that are linked to renal Fanconi syndrome. These findings provide new perspectives for the treatment of nephropathic cystinosis and other renal lysosomal storage diseases.Background Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis.Methods To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish.Results Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy?lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin.Conclusions Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.