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

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.

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Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

International Journal of Molecular Sciences ◽

10.3390/ijms21010192 ◽

2019 ◽

Vol 21 (1) ◽

pp. 192 ◽

Cited By ~ 6

Author(s):

Domenico De Rasmo ◽

Anna Signorile ◽

Ester De Leo ◽

Elena V. Polishchuk ◽

Anna Ferretta ◽

...

Keyword(s):

Fanconi Syndrome ◽

Mitochondrial Dynamics ◽

Proteolytic Processing ◽

Nephropathic Cystinosis ◽

Lysosomal Storage ◽

Mitochondrial Fragmentation ◽

Mitofusin 2 ◽

Size Number ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular

Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in CTNS gene leading to Fanconi syndrome. Independent studies reported defective clearance of damaged mitochondria and mitochondrial fragmentation in cystinosis. Proteins involved in the mitochondrial dynamics and the mitochondrial ultrastructure were analyzed in CTNS−/− cells treated with cysteamine, the only drug currently used in the therapy for cystinosis but ineffective to treat Fanconi syndrome. CTNS−/− cells showed an overexpression of parkin associated with deregulation of ubiquitination of mitofusin 2 and fission 1 proteins, an altered proteolytic processing of optic atrophy 1 (OPA1), and a decreased OPA1 oligomerization. According to molecular findings, the analysis of electron microscopy images showed a decrease of mitochondrial cristae number and an increase of cristae lumen and cristae junction width. Cysteamine treatment restored the fission 1 ubiquitination, the mitochondrial size, number and lumen of cristae, but had no effect on cristae junction width, making CTNS−/− tubular cells more susceptible to apoptotic stimuli.

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Protection of Cystinotic Mice by Kidney-Specific Megalin Ablation Supports an Endocytosis-Based Mechanism for Nephropathic Cystinosis Progression

Journal of the American Society of Nephrology ◽

10.1681/asn.2019040371 ◽

2019 ◽

Vol 30 (11) ◽

pp. 2177-2190 ◽

Cited By ~ 1

Author(s):

Virginie Janssens ◽

Héloïse P. Gaide Chevronnay ◽

Sandrine Marie ◽

Marie-Françoise Vincent ◽

Patrick Van Der Smissen ◽

...

Keyword(s):

Proximal Tubular Cell ◽

Tubular Cell ◽

Nephropathic Cystinosis ◽

Lysosomal Storage ◽

Proximal Tubular Cells ◽

Renal Fanconi Syndrome ◽

Crystal Deposition ◽

Tubular Cells ◽

Proximal Tubular ◽

Kidney Proximal Tubular Cells

BackgroundDeletions or inactivating mutations of the cystinosin gene CTNS lead to cystine accumulation and crystals at acidic pH in patients with nephropathic cystinosis, a rare lysosomal storage disease and the main cause of hereditary renal Fanconi syndrome. Early use of oral cysteamine to prevent cystine accumulation slows progression of nephropathic cystinosis but it is a demanding treatment and not a cure. The source of cystine accumulating in kidney proximal tubular cells and cystine’s role in disease progression are unknown.MethodsTo investigate whether receptor-mediated endocytosis by the megalin/LRP2 pathway of ultrafiltrated, disulfide-rich plasma proteins could be a source of cystine in proximal tubular cells, we used a mouse model of cystinosis in which conditional excision of floxed megalin/LRP2 alleles in proximal tubular cells of cystinotic mice was achieved by a Cre-LoxP strategy using Wnt4-CRE. We evaluated mice aged 6–9 months for kidney cystine levels and crystals; histopathology, with emphasis on swan-neck lesions and proximal-tubular-cell apoptosis and proliferation (turnover); and proximal-tubular-cell expression of the major apical transporters sodium-phosphate cotransporter 2A (NaPi-IIa) and sodium-glucose cotransporter-2 (SGLT-2).ResultsWnt4-CRE–driven megalin/LRP2 ablation in cystinotic mice efficiently blocked kidney cystine accumulation, thereby preventing lysosomal deformations and crystal deposition in proximal tubular cells. Swan-neck lesions were largely prevented and proximal-tubular-cell turnover was normalized. Apical expression of the two cotransporters was also preserved.ConclusionsThese observations support a key role of the megalin/LRP2 pathway in the progression of nephropathic cystinosis and provide a proof of concept for the pathway as a therapeutic target.

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Approach to the patient with renal Fanconi syndrome, glycosuria, or aminoaciduria

10.1093/med/9780199592548.003.0041_update_001 ◽

2018 ◽

Author(s):

Detlef Bockenhauer ◽

Robert Kleta

Keyword(s):

Fanconi Syndrome ◽

Tubular Dysfunction ◽

Renal Fanconi Syndrome ◽

Renal Glycosuria ◽

Proximal Tubular Dysfunction ◽

Wide Range ◽

Proximal Tubular ◽

Proximal Tubular Function ◽

Low Molecular Weight Proteins ◽

Filtration Capacity

Up to 80% of filtered salt and water is returned back into the circulation in the proximal tubule. Several solutes, such as phosphate, glucose, low-molecular weight proteins, and amino acids are exclusively reabsorbed in this segment, so their appearance in urine is a sign of proximal tubular dysfunction. An entire orchestra of specialized apical and basolateral transporters, as well as paracellular molecules, mediate this reabsorption. Defects in proximal tubular function can be isolated (e.g. isolated renal glycosuria, aminoacidurias, or hypophosphataemic rickets) or generalized. In the latter case it is called the Fanconi–Debre–de Toni syndrome, based on the initial clinical descriptions. However, in clinical practice it is usually referred to as just the ‘renal Fanconi syndrome’. Severity of proximal tubular dysfunction can vary, and may coexist with some degree of loss of glomerular filtration capacity. Causes include a wide range of insults to proximal tubular cells, including a number of genetic conditions, drugs and poisons.

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Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure

Journal of the American Society of Nephrology ◽

10.1681/asn.2017111179 ◽

2018 ◽

Vol 29 (7) ◽

pp. 1849-1858 ◽

Cited By ~ 13

Author(s):

Markus Reichold ◽

Enriko D. Klootwijk ◽

Joerg Reinders ◽

Edgar A. Otto ◽

Mario Milani ◽

...

Keyword(s):

Kidney Failure ◽

Fanconi Syndrome ◽

Genetic Disorder ◽

Subsequent Development ◽

Mitochondrial Morphology ◽

Missense Mutations ◽

Renal Fanconi Syndrome ◽

Biopsy Specimens ◽

Proximal Tubular ◽

Renal Proximal Tubular

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

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Nephropathic Cystinosis: A Distinct Form of CKD–Mineral and Bone Disorder that Provides Novel Insights into the Regulation of FGF23

Journal of the American Society of Nephrology ◽

10.1681/asn.2019111172 ◽

2020 ◽

Vol 31 (9) ◽

pp. 2184-2192

Author(s):

Pablo Florenzano ◽

Macarena Jimenez ◽

Carlos R. Ferreira ◽

Galina Nesterova ◽

Mary Scott Roberts ◽

...

Keyword(s):

Fibroblast Growth Factor 23 ◽

Serum Phosphate ◽

Nephropathic Cystinosis ◽

Lysosomal Storage ◽

Mineral And Bone Disorder ◽

Renal Fanconi Syndrome ◽

Mineral Homeostasis ◽

Ckd Stage ◽

Common Causes ◽

Bone Disorder

BackgroundThe rare lysosomal storage disease nephropathic cystinosis presents with renal Fanconi syndrome that evolves in time to CKD. Although biochemical abnormalities in common causes of CKD–mineral and bone disorder have been defined, it is unknown if persistent phosphate wasting in nephropathic cystinosis is associated with a biochemical mineral pattern distinct from that typically observed in CKD–mineral and bone disorder.MethodsWe assessed and compared determinants of mineral homeostasis in patients with nephropathic cystinosis across the predialysis CKD spectrum to these determinants in age- and CKD stage-matched patients, with causes of CKD other than nephropathic cystinosis.ResultsThe study included 50 patients with nephropathic cystinosis-related CDK and 97 with CKD from other causes. All major aspects of mineral homeostasis were differentially effected in patients with CKD stemming from nephropathic cystinosis versus other causes. Patients with nephropathic cystinosis had significantly lower percent tubular reabsorption of phosphate and fibroblast growth factor-23 (FGF23) at all CKD stages, and lower blood phosphate in CKD stages 3–5. Linear regression analyses demonstrated lower FGF23 levels in nephropathic cystinosis participants at all CKD stages when corrected for eGFR and age, but not when adjusted for serum phosphate.ConclusionsNephropathic cystinosis CKD patients have mineral abnormalities that are distinct from those in CKD stemming from other causes. Persistently increased urinary phosphate excretion maintains serum phosphate levels within the normal range, thus protecting patients with nephropathic cystinosis from elevations of FGF23 during early CKD stages. These findings support the notion that phosphate is a significant driver of increased FGF23 levels in CKD and that mineral abnormalities associated with CKD are likely to vary depending on the underlying renal disease.

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

International Journal of Molecular Sciences ◽

10.3390/ijms222312829 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12829

Author(s):

Francesco Bellomo ◽

Ester De Leo ◽

Anna Taranta ◽

Laura Giaquinto ◽

Gianna Di Giovamberardino ◽

...

Keyword(s):

Gene Expression ◽

Rare Diseases ◽

Gene Expression Signature ◽

Drug Repurposing ◽

Lysosomal Storage Diseases ◽

Nephropathic Cystinosis ◽

Lysosomal Storage ◽

Expression Signature ◽

Integrative Study ◽

Development Processes

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

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Evaluation of the proximal tubular function in hereditary renal Fanconi syndrome

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfn373 ◽

2008 ◽

Vol 23 (9) ◽

pp. 2719-2722 ◽

Cited By ~ 7

Author(s):

L. Monnens ◽

E. Levtchenko

Keyword(s):

Fanconi Syndrome ◽

Tubular Function ◽

Renal Fanconi Syndrome ◽

Proximal Tubular ◽

Proximal Tubular Function

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Clinical Heterogeneity and Phenotypic Expansion of NaPi-IIa–Associated Disease

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2017-01592 ◽

2017 ◽

Vol 102 (12) ◽

pp. 4604-4614 ◽

Cited By ~ 11

Author(s):

Korcan Demir ◽

Melek Yıldız ◽

Hilla Bahat ◽

Michael Goldman ◽

Nisreen Hassan ◽

...

Keyword(s):

Fanconi Syndrome ◽

Loss Of Function ◽

Renal Fanconi Syndrome ◽

Whole Exome ◽

Proximal Tubular ◽

History Of ◽

Proximal Tubulopathy ◽

Idiopathic Infantile Hypercalcemia ◽

Close Relatives

Abstract Context NaPi-IIa, encoded by SLC34A1, is a key phosphate transporter in the mammalian proximal tubule and plays a cardinal role in renal phosphate handling. NaPi-IIa impairment has been linked to various overlapping clinical syndromes, including hypophosphatemic nephrolithiasis with osteoporosis, renal Fanconi syndrome with chronic kidney disease, and, most recently, idiopathic infantile hypercalcemia and nephrocalcinosis. Objectives We studied the molecular basis of idiopathic infantile hypercalcemia with partial proximal tubulopathy in two apparently unrelated patients of Israeli and Turkish descent. Design Genetic analysis in two affected children and their close relatives was performed using whole-exome sequencing, followed by in vitro localization and trafficking analysis of mutant NaPi-IIa. Results Mutation and haplotype analyses in both patients revealed a previously described homozygous loss-of-function inserted duplication (p.I154_V160dup) in NaPi-IIa, which is inherited identical-by-descent from a common ancestor. The shared mutation was originally reported by our team in two adult siblings with renal Fanconi syndrome, hypophosphatemic bone disease, and progressive renal failure who are family members of one of the infants reported herein. In vitro localization assays and biochemical analysis of p.I154_V160dup and of additional NaPi-IIa mutants harboring a trafficking defect indicate aberrant retention at the endoplasmic reticulum in an immature and underglycosylated state, leading to premature proteasomal degradation. Conclusions Our findings expand the phenotypic spectrum of NaPi-IIa disruption, reinforce its link with proximal tubular impairment, enable longitudinal study of the natural history of the disease, and shed light on cellular pathways associated with loss of function and impaired trafficking of NaPi-IIa mutants.

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