Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype

Abstract

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures.

Co-axial printing of convoluted proximal tubule for kidney disease modeling

“…Mutations in this gene result in nephropathic cystinosis, a chronic condition in which lysosomal accumulation of cystine leads to loss of PT integrity (figure 1(A)). Furthermore, it has been demonstrated that a 3D membrane-based model of ciPTEC CTNS -/recapitulated better the nephropathic phenotype at a molecular, structural, and functional PT level compared to 2D cell cultures [10]. This observation highlights the importance of using a biomimetic microenvironment to recapitulate kidney functions.…”

“…Tight sealing of our engineered chip was confirmed by perfusing colored MilliQ water through a microfiber not containing cells (supplementary movie 1). It has been previously demonstrated that CTNS -/cells grown on hollow fiber membranes form a less tight monolayer in comparison to a healthy control [10]. Here, we evaluated cell monolayer tightness by means of an inulin-FITC leakage assay.…”

“…The ciPTEC 14.4 line used for this work has shown to stably express the PT phenotype over a very high number of passages and when grown as kidney tubules on a fiber [10,21,22]. Further, the cystinotic cell line (ciPTEC CTNS -/-) showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and disruption of the epithelial monolayer tightness as compared to control kidney tubules [10]. These features are representative of the nephropathic phenotype.…”

CTNSmRNA as a potential treatment for nephropathic cystinosis

“…4,21 The hollow fiber membrane (HFM) 3D model is representative of a functional kidney tubule and was prepared by seeding PTECs on L-DOPA and Collagen-IV coated fibers at 33°C, as described. 22 Experiments were performed after differentiation at 37°C for 10 (PTECs) or 12 days (PODOs).…”

“…HFM were also stained for the 3HA-tag, F-actin and LAMP1, according to previously described protocols. 22 Cystine measurement in vitro Cystine levels were measured, as described previously, 21 in a total volume of 100µL 50mM Nethylmaleimide (Sigma, cat.no.04259) and 50µL of 12% sulfosalicylic acid (Sigma, cat.no.S7422).…”