Renal proximal tubular epithelium from patients with nephropathic cystinosis: Immortalized cell lines as in vitro model systems

Renal proximal tubular epithelium from patients with nephropathic cystinosis: Immortalized cell lines as in vitro model systems. The renal proximal tubule is a major site of injury in a variety of congenital/metabolic diseases including nephropathic cystinosis, the most commonly known cause of renal Fanconi's syndrome. In this lysosomal storage disease there are defects in proximal tubule function within the first few months of life. While culture of renal tubular cells from the urine of these patients is possible, development of immortalized cell lines would insure large numbers of homogeneous cells for studies of renal epithelial cell morphology and pathophysiology in this disease. To develop immortalized cells, cystinotic and normal proximal tubular cells in culture were exposed to an immortalizing vector, containing pZiptsU19 with the temperature sensitive SV40 T-antigen allele tsA58U19 and a neomycin resistance gene, and neomycin-resistant tubular cells were selected for propagation. Ten clones from cystinotic patients have been developed and characterized. All clones express T-antigen at permissive temperature (33°C). Immortalized cells have an epithelial morphology and grow to form confluent monolayers; doubling times vary from 31 to 86 hours. Cystinotic clones are keratin, MDR P-glycoprotein, and alpha-95 kD brush-border associated protein positive but Tamm-Horsfall protein negative by immunocytochemistry, as are normal proximal tubule cells immortalized with this vector. This is consistent with a proximal tubule origin of the cystinotic clones. The cystine content of the cystinotic cells is 70 to 160 times that of normal renal proximal tubular cells in culture, with most of the cystine sequestered in cell lysosomes, confirming that these cell lines express the storage defect. Lysosomes from representative cell lines give a dense lysosome fraction upon Percoll gradient centrifugation, making it possible to purify lysosomes from these cells with no significant contamination by other organelles. These immortalized cystinotic human renal tubular epithelial cell lines should serve as a useful in vitro model for the study of tubular epithelial cell and lysosomal abnormalities in cystinosis, enabling definition of pathogenesis of tubular cell dysfunction in this prototypic disease