Urine-Derived Kidney Progenitor Cells in Cystinosis

Abstract

Nephropathic cystinosis is an inherited lysosomal storage disorder caused by pathogenic variants in the cystinosin (CTNS) gene and is characterized by the excessive shedding of proximal tubular epithelial cells (PTECs) and podocytes into urine, development of the renal Fanconi syndrome and end-stage kidney disease (ESKD). We hypothesized that in compensation for epithelial cell losses, cystinosis kidneys undertake a regenerative effort, and searched for the presence of kidney progenitor cells (KPCs) in the urine of cystinosis patients. Urine was cultured in a specific progenitor medium to isolate undifferentiated cells. Of these, clones were characterized by qPCR, subjected to a differentiation protocol to PTECs and podocytes and assessed by qPCR, Western blot, immunostainings and functional assays. Cystinosis patients voided high numbers of undifferentiated cells in urine, of which various clonal cell lines showed a high capacity for self-renewal and expressed kidney progenitor markers, which therefore were assigned as cystinosis urine-derived KPCs (Cys-uKPCs). Cys-uKPC clones showed the capacity to differentiate between functional PTECs and/or podocytes. Gene addition with wild-type CTNS using lentiviral vector technology resulted in significant reductions in cystine levels. We conclude that KPCs present in the urine of cystinosis patients can be isolated, differentiated and complemented with CTNS in vitro, serving as a novel tool for disease modeling.

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis

“…Conditionally immortalized proximal tubular epithelial cells (PTECs) and podocytes (PODOs), derived from healthy donors (WT) and cystinosis (CYS) patients (homozygous 57 kb deletion) 3 , 44 – 46 , were cultured in DMEM F-12 (Biowest), as described before 4 , 47 . 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 17 .…”

“…Cystine levels were measured, as described previously 47 . Cells were lysed in 100 µl 50 mM N -ethylmaleimide (NEM-Sigma) and 50 µl of 12% sulfosalicylic acid (SSA-Sigma).…”

CTNSmRNA as a potential treatment for nephropathic cystinosis

“…Conditionally immortalized proximal tubular epithelial cells (PTECs) and podocytes (PODOs), derived from healthy donors (WT) and cystinosis (CYS) patients (57kb deletion), 3,[18][19][20] were cultured in DMEM F-12 (Biowest, cat.no.L0093-500), as described before. 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).…”

Renal progenitors derived from urine for personalized diagnosis of kidney diseases

“…These cells exhibit a limited expandability and lifespan due to their mature differentiation state, requiring immortalization to maintain them in vitro [5‒10]. Immortalized urinary podocytes and tubular cells of healthy volunteers and patients established by hTERT, temperature-sensitive SV40 T antigen transfection or the introduction of viral oncogenes possess strong proliferative potential and could acquire the phenotype of fully differentiated cells in response to changes in culture conditions (such as temperature, exposure to retinoic acid, retinoic acid + vitamin D3, hydrocortisone + EGF + tri-iodothyronine) [9, 11‒15]. Even if several studies highlight the usefulness of these immortalized-kidney cells for disease modeling [6, 12, 16, 17], drug screening [12, 14], and bioengineering application [13, 18], the invasive procedure of immortalization could affect the transcriptome and interfere with cellular pathways of cells, which might lose their sensitivity to external stimuli [5, 19].…”