Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis

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.