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

Abstract

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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Kinase X Activates Ureteric Bud Branching Morphogenesis in Developing Mouse Metanephric Kidney

“…In situ hybridization using the digoxigenin-substituted 220-bp PRKX PCR fragment as probe (Figure 2A through D) showed that PRKX mRNA transcripts were specifically local- ized throughout the ureteric bud epithelial cells of 14-to 17-wk human fetal kidneys from the medullary truncal regions through to the cortical branching tips, which also expressed the characteristic ureteric bud epithelial markers cytokeratin ( Figure 2E) and polycystin-1 ( Figure 2F) (24,25). PRKX mRNA and protein are also highly expressed in primary and differentiated conditionally immortalized human renal HFCT epithelial cell lines derived from microdissected fetal kidneys (Figure 3, A and B).…”

“…Briefly, microdissected human fetal (19 wk) collecting tubules (HFCT) were conditionally immortalized using a temperature-sensitive T antigen expressing retroviral vector (25) and clonal cells lines derived by limiting dilution. For studies described here, the HFCT clone 7F was grown to 70% confluence at 33°C then transferred to the nonpermissive temperature of 37°C for 7 to 10 d before use in adhesion and migration assays.…”

Role of Ubiquitin-Like Protein FAT10 in Epithelial Apoptosis in Renal Disease

“…To conditionally immortalize proximal tubular epithelial cells (PTEC), we developed a lentiviral vector encoding the tsTag allele tsA58U19 (20). tsTag was subcloned into pIRES-hyg (Clontech, Palo Alto, CA).…”

Ocrl1, a PtdIns(4,5)P25-Phosphatase, Is Localized to theTrans-Golgi Network of Fibroblasts and Epithelial Cells

“…A normal human kidney epithelial cell line (NHK52) bearing a temperature-sensitive SV40 large T-antigen plasmid was provided by Dr. L.C. Racusen (Racusen et al 1995). NHK cells were maintained in media containing 50% Ham's F12, 50% Dulbecco's modified Eagles' medium, 5% fetal bovine serum, 25 mM Hepes, 2.5 g/ml insulin, 50 ng/ml hydrocortisone, 5 g/ml transferrin, 5 ng/ml sodium selenite, 20 ng/ml triiodothyronine, 50 U/ml penicillin, 50 g/ml streptomycin, and 2.5 g/ml amphotericin.…”