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 co ngenital/metabolic diseases including nephropathic cystinosis, the mos t commonly known cause of renal Fanconi's syndrome. In this lysosomal storage disease there are defects in proximal tubule function within t he 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 disea se. To develop immortalized cells, cystinotic and normal proximal tubu lar cells in culture were exposed to an immortalizing vector, containi ng pZiptsU19 with the temperature sensitive SV40 T-antigen allele tsA5 8U19 and a neomycin resistance gene, and neomycin-resistant tubular ce lls were selected for propagation. Ten clones from cystinotic patients have been developed and characterized. All clones express T-antigen a t permissive temperature (33 degrees C). Immortalized cells have an ep ithelial morphology and grow to form confluent monolayers; doubling ti mes vary from 31 to 86 hours. Cystinotic clones are keratin, MDR P-gly coprotein, and alpha-95 kD brush-border associated protein positive bu t Tamm-Horsfall protein negative by immunocytochemistry, as are normal proximal tubule cells immortalized with this vector. This is consiste nt with a proximal tubule origin of the cystinotic clones. The cystine content of the cystinotic cells is 70 to 160 times that of normal ren al proximal tubular cells in culture, with most of the cystine sequest ered in cell lysosomes, confirming that these eel lines express the st orage defect. Lysosomes from representative cell lines give a dense ly sosome fraction upon Percoll gradient centrifugation, making it possib le to purify lysosomes from these cells with no significant contaminat ion by other organelles. These immortalized cystinotic human renal tub ular epithelial cell lines should serve as a useful in vitro model for the study of tubular epithelial cell and lysosomal abnormalities in c ystinosis, enabling definition of pathogenesis of tubular cell dysfunc tion in this prototypic disease.