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

Abstract

BackgroundMutations 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.MethodsTo 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.ResultsOf 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.ConclusionsOur 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.

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

“…Transport characteristics of the ABCG2 and MRP4 renal efflux transporter can be assessed by liquid scintillation counting or imaging after incubating the cells with labeled kynurenic acid or with Chloromethylfluorescein-diacetate (CMFDA) respectively and an MRP inhibitor. GST-RAP or the transferrin endocytosis assay can be utilized to study the functionality of the megalin-cubilin receptor complex by analyzing the endocytosis process itself [102][103][104][105][106][107].…”

“…Of the 46 compounds significantly reducing p62/SQSTM1 levels in cystinotic cells, they chose luteolin to study further based on its efficacy and safety profiles. In their study, luteolin had anti-apoptotic properties, was a powerful antioxidant and could stimulate endocytosis through enhancing the expression of the endocytic receptor megalin, which are all beneficial effects for the cystinotic kidney [107].…”

“…Although the numbers of urine-derived podocyte and PTEC models developed to study genetic kidney diseases are rapidly increasing over the last few years, most diseases affecting these cells still lack representative urine-derived models [6,8,100,107]. With the new era of personalized medicine in genetic kidney diseases, not only uncovering the genetic background and linking this genotype to its clinical phenotype is required, but also investigating the individualized response of each patient to various management strategies, in what is known as personalized therapy [17,212,213].…”

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis

“…Gene-expression signatures of compounds identified by drug screening were combined with the transcriptional profile of the disease of interest. Our disease model was nephropathic cystinosis, a rare genetic disorder characterized by cystine accumulation [ 36 ], tendency of cells to undergo apoptosis [ 37 , 38 ], mitochondrial impairment [ 39 , 40 ], and defective autophagy [ 41 , 42 ], among others. The current therapy for cystinosis is limited to cysteamine.…”

“…Alexidine dihydrochloride was shown to have the most potent lysosomal cholesterol-reducing activity in a high-content screening for modifiers of Niemann-Pick type C disease [ 47 ] and it was also a hit of the previous cell-based phenotypic drug screening for compounds that reduced the autophagy-related protein p62/SQSTM1 levels in cystinotic cells [ 42 ]. Beta-escin has an antioxidant potential [ 48 ] and induces perturbation in cholesterol homeostasis, which causes a cascade of cellular responses like decreased NFκB activation [ 14 ].…”