Cystinosin-deficient rats recapitulate the phenotype of nephropathic cystinosis

Hollywood, Jennifer A.; Kallingappa, Prasanna; Cheung, Pang Yuk; Martis, Renita M; Sreebhavan, Sree; Atiola, Robert D.; Chatterjee, A. K.; Buckels, Emma J.; Matthews, Brya G.; Lewis, Paula; Davidson, Alan J.

doi:10.1152/ajprenal.00277.2021

Cited by 5 publications

(1 citation statement)

References 49 publications

(53 reference statements)

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“…To assess and cross-validate the structural and functional impact of these lysosomal changes in human cystinotic RPTECs we generated a CTNS -/- in vitro, HuIm RPTEC cystinosis model (16, 46). We found an association between the knocking out of the CTNS and the downregulation of v-ATPases.…”

Section: Discussionmentioning

confidence: 99%

Novel Mechanism for Tubular Injury in Nephropathic Cystinosis

Sur

Kerwin

Pineda

et al. 2022

Preprint

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Understanding the unique susceptibility of the human kidney to pH dysfunction and injury in cystinosis is paramount to develop new therapies to preserve renal function. Renal proximal tubular epithelial cells (RPTECs) and fibroblasts, isolated from patients with cystinosis were transcriptionally profiled. Lysosomal fractionation, immunoblotting, confocal microscopy, intracellular pH, TEM, mitochondrial stress test, and membrane integrity assays were performed for validation. CRISPR, CTNS-/- RPTECs were generated. A new compound, Astaxanthin (ATX), was evaluated for rescue of the CTNS-/- phenotype. Alterations in cell stress, pH, autophagic turnover, and mitochondrial energetics, highlighted key changes in the vacuolar (V)-ATPases in patient derived and CTNS-/- RPTECs. ATP6V0A1 was significantly downregulated in cystinosis and highly co-regulated with loss of CTNS. Overexpression of ATP6V0A1 rescued cell stress and mitochondrial function. Treatment of CTNS-/- RPTECs with ATX, induced ATP6V0A1 expression and the resulting rescue of the RPTE cell and mitochondrial injury.In conclusion, our exploratory transcriptional and in vitro cellular and functional studies confirm that loss of cystinosin in RPTEC, results in a reduction in ATP6V0A1 expression, with changes in intracellular pH, mitochondrial integrity, mitochondrial function, and autophagosome-lysosome clearance. ATX can rescue the cystinotic RPTEC injury, at least partially mediated by upregulating ATP6V0A1 expression. The availability of ATX as a well-tolerated oral supplement, offers its further clinical evaluation as a potential novel therapeutic to limit renal tubular injury in cystinosis, independent of cysteine depletion alone.

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Section: Discussionmentioning

confidence: 99%

Novel Mechanism for Tubular Injury in Nephropathic Cystinosis

Sur

Kerwin

Pineda

et al. 2022

Preprint

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Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content

Rega,

Janssens,

Graversen

et al. 2023

Sci Rep

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Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by l- and d-stereoisomers. In cystinotic mice, 2-month diets with 5x-l-lysine and 5x-l-arginine were overall well tolerated, while 5x-d-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-d-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by d-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

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MFSD12 depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis

Bondue,

Khodaparast,

Khodaparast

et al. 2024

American Journal of Physiology-Renal Physiology

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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 to the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC ( CTNS Δ 57kb) 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. Additionally, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development, warrants further evaluation.

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Cystinosin-deficient rats recapitulate the phenotype of nephropathic cystinosis

Cited by 5 publications

References 49 publications

Novel Mechanism for Tubular Injury in Nephropathic Cystinosis

Novel Mechanism for Tubular Injury in Nephropathic Cystinosis

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content

MFSD12 depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis

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