The protein product of the gene that when mutated is responsible for Lowe syndrome, or oculocerebrorenal syndrome (OCRL), is an inositol polyphosphate 5-phosphatase. It has a marked preference for phosphatidylinositol 4,5-bisphosphate although it hydrolyzes all four of the known inositol polyphosphate 5-phosphatase substrates: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The enzyme activity of this protein is determined by a region of 672 out of a total of 970 amino acids that is homologous to inositol polyphosphate 5-phosphatase II. Cell lines from kidney proximal tubules of a patient with Lowe syndrome and a normal individual were used to study the function of OCRL. The cells from the Lowe syndrome patient lack OCRL protein. OCRL is the major phosphatidylinositol 4,5-bisphosphate 5-phosphatase in these cells. As a result, these cells accumulate phosphatidylinositol 4,5-bisphosphate even though at least four other inositol polyphosphate 5-phosphatase isozymes are present in these cells. OCRL is associated with lysosomal membranes in control proximal tubule cell lines suggesting that OCRL may function in lysosomal membrane trafficking by regulating the specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes.Lowe syndrome, or oculocerebrorenal syndrome, is a rare X chromosome-linked disorder that is characterized by severe mental retardation, congenital cataracts, and renal Fanconi syndrome (1). The renal Fanconi syndrome develops in the neonatal period with impaired renal proximal tubular function including acidosis, amino aciduria, phosphaturia, and proteinuria (1, 2). The gene responsible for Lowe syndrome was identified by positional cloning of X chromosome breakpoints in two affected females (3). The predicted protein, designated OCRL, 1 is comprised of 970 amino acids and is 51% identical to inositol polyphosphate 5-phosphatase type II (5-phosphatase II) over a span of 672 amino acids. The amino-terminal one-third has no homology to 5-phosphatase II or any other sequences in GenBank (3). The striking homology between OCRL and 5-phosphatase II suggested that OCRL belongs to the 5-phosphatase gene family and that Lowe syndrome represents an inborn error of inositol phosphate metabolism.Inositol polyphosphate 5-phosphatases (5-phosphatases) are a group of enzymes containing 5-phosphatase homology domains, and two conserved signature motifs within these domains define proteins that have 5-phosphatase activity (for review, see Refs. 4 -6). The substrates of 5-phosphatases include two soluble inositol polyphosphates inositol 1,4,5-trisphosphate (Ins 1,4,5-P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P 4 ), and two inositol lipids phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ). The 5-phosphatase isozymes have varying substrate specificities. Seven mammalian 5-phosphatases have been cloned and...

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)

The mechanisms of toxicity of cyclosporine A (CsA) were studied in primary cultures of individually microdissected rabbit and human renal tubules of proximal and distal regions of the nephron. A direct toxic effect of CsA on renal tubule epithelia was demonstrated using nigrosine uptake and LDH release as indicators of cell death. Proximal convoluted tubules (PCT) and proximal straight tubules (PST) were shown to be highly sensitive, while thick ascending limbs of Henle (TAL) were much less sensitive and cortical collecting tubules (CCT) relatively resistant. The effects of CsA were time and dose dependent over the range 50 ng/ml to 100 micrograms/ml. Protection against CsA-induced PST cell death was afforded by reduction in extracellular calcium levels in the media or addition of the calcium entry antagonists: verapamil, nifedipine or diltiazem. In addition, treatment with the cysteine protease inhibitor, E64, attenuated CsA-induced cell damage. A role for the lysosomal cysteine proteases (cathepsins), however, was ruled out on the basis of identical activity levels in all cell types; no beneficial effects of lysosomal enzyme depletion and no evidence of lysosomal rupture prior to death. By contrast, a role for the cytoplasmic, calcium-dependent cysteine protease calpain was suggested since activity levels were significantly higher in PST than CCT cultures and were inducible by CsA.

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