Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype.

Elena Sendino Garví,Manoe J Janssen,Rosalinde Masereeuw

doi:10.3390/cells11010177

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.

Highlights

Nephropathic cystinosis is an autosomal recessive chronic kidney disease condition caused by mutations in the CTNS gene [1,2]
One of the first manifestations of cystinosis is the clinical presentation of renal Fanconi syndrome, characterized by a severe proximal tubule cell dysfunction at early stages of the disease, which results in a total loss of integrity of the proximal tubule [7]
We used the conditionally immortalized proximal tubule epithelial cells (ciPTECs) CTNSWT cells as our healthy control line; these cells have been well characterized in the past and are known to maintain many proximal tubules cell functions in culture [24]

Summary

Introduction

Nephropathic cystinosis is an autosomal recessive chronic kidney disease condition caused by mutations in the CTNS gene [1,2]. Several mutations have been associated with this disease, but the most recurrent in Europe is a 57kb deletion including the first 10 exons of the CTNS gene [3]. This gene encodes for a cystine/proton symporter located in the lysosomal membrane. The impairment or loss of cystinosin leads to the accumulation of cystine inside the lysosomes in all the cells of the body [4–6], which causes severe and chronic damage to several organs, the kidneys. Great efforts have been made to elucidate further the underlying pathological mechanisms of nephropathic cystinosis that revealed several hallmarks beyond cystine accumulation, including impaired autophagy, mTOR activation, disrupted vesicle dynamics (lysosomes-autophagosomes interactions), mitochondrial impairment, reactive oxygen species (ROS), and increased cell stress [8–13]

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