Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.

Distal renal tubular acidosis in Filipino children, caused by mutations of the anion-exchanger SLC4A1 (AE1, Band 3) gene.

AIM: To describe the clinical features and genetic basis of distal renal tubular acidosis (dRTA) in Filipino children. METHODS: Clinical description and gene analysis of affected members of 7 families. RESULTS: In all affected children, the disease was associated with mutations of the SLC4A1 gene that codes for the bicarbonate/chloride anion-exchanger 1 (AE1, band 3) protein situated in the red cell membrane and the alpha-intercalated (proton-secreting) cell of the renal collecting duct. In 2 families, affected children were homozygous for a substitution of aspartic acid for glycine in residue 701 of the AE1 protein (G701D); in the other 5 families, affected children were compound heterozygotes of this mutation with the AE1 mutation (Delta400-408) that causes Southeast Asian ovalocytosis (SAO). All affected children had morphological red cell changes that closely resembled SAO, including the children who were homozygous for G701D and did not have the SAO mutation. Homozygous G701D thus produces morphological red cell changes that are not readily distinguishable from SAO. The parents of all 7 families were originally domiciled in the islands of the Visayas group in the central part of the Philippine archipelago. CONCLUSION: Recessive renal tubular acidosis in Filipinos is usually caused by SLC4A1 mutations, commonly G701D.

OCRL1 mutations in Dent 2 patients suggest a mechanism for phenotypic variability.

BACKGROUND/AIMS: Dent disease is an X-linked renal proximal tubulopathy associated with mutations in CLCN5 (Dent 1) or OCRL1 (Dent 2). OCRL1 mutations also cause the oculocerebrorenal syndrome of Lowe. METHODS: Dent patients with normal sequence for CLCN5 were sequenced for mutations in OCRL1. By analyzing these and all other OCRL1 mutations reported, a model relating OCRL1 mutations to the resulting disease (Dent 2 or Lowe's) was developed. RESULTS: Six boys with Dent disease had novel OCRL1 mutations: two missense (R301H, G304E) and four mutations predicted to produce premature termination codons (L56DfsX1, S149X, P161PfsX3, and M170IfsX1). These include one of the original patients reported by Dent and Friedman. Slit lamp examinations revealed early cataracts in only one boy with normal vision. None of these Dent 2 patients had metabolic acidosis; 3 had mild mental retardation. Analysis of all known OCRL1 mutations show that Dent 2 mutations fall into two classes that do not overlap with Lowe mutations. Bioinformatics analyses identified expressed OCRL1 splice variants that help explain the variability of those clinical features that distinguish Dent disease from Lowe syndrome. CONCLUSIONS: OCRL1 mutations can cause the renal phenotype of Dent disease, without acidosis or the dramatic eye abnormalities typical of Lowe syndrome. We propose a model to explain the phenotypic variability between Dent 2 and Lowe's based on distinctly different classes of mutations in OCRL1 producing splice variants.

Sevelamer and other anion-exchange resins in the prevention and treatment of hyperphosphataemia in chronic renal failure.

Sevelamer, or more precisely 'sevelamer hydrochloride', is a weakly basic anion-exchange resin in the chloride form that was introduced in 1997 for the treatment of the hyperphosphataemia of patients with end-stage renal failure, usually those on long-term haemodialysis. The rationale for this therapy was that sevelamer would sequester phosphate within the gastrointestinal tract, so preventing its absorption and enhancing its faecal excretion. Over the succeeding years, large numbers of patients have been treated with sevelamer, and it has fulfilled expectations in helping to control the hyperphosphataemia of end-stage renal failure. However, it is only one of many anion-exchange resins that could be used for this purpose, some of which are currently available for clinical use and are much less costly than sevelamer. Theoretical considerations suggest that some of these other resins might be at least as efficient as sevelamer in sequestering phosphate in the gastrointestinal tract. Neither sevelamer, nor any of these other agents, has been submitted to a proper metabolic balance study to measure the amount of phosphate sequestered by the resin in the bowel, and without this information it is impossible to judge which is the ideal resin for this purpose.

Distal renal tubular acidosis: alkali heals osteomalacia and increases net production of 1,25-dihydroxyvitamin D.

In 2 women with distal renal tubular acidosis and osteomalacia, alkali treatment cured the bone disease and was accompanied by marked increases in the serum 1,25 dihydroxyvitamin D concentration, without a significant change in the 25-hydroxyvitamin D concentration.

Recessive distal renal tubular acidosis in Sarawak caused by AE1 mutations.

Mutations of the AE1 (SLC4A1, Anion-Exchanger 1) gene that codes for band 3, the renal and red cell anion exchanger, are responsible for many cases of familial distal renal tubular acidosis (dRTA). In Southeast Asia this disease is usually recessive, caused either by homozygosity of a single AE1 mutation or by compound heterozygosity of two different AE1 mutations. We describe two unrelated boys in Sarawak with dRTA associated with compound heterozygosity of AE1 mutations. Both had Southeast Asian ovalocytosis (SAO), a morphological abnormality of red cells caused by a deletion of band 3 residues 400-408. In addition, one boy had a DNA sequence abnormality of band 3 residue (G701D), which has been reported from elsewhere in Southeast Asia. The other boy had the novel sequence abnormality of band 3 (Q759H) and profound hemolytic anemia.

Band 3 Walton, a C-terminal deletion associated with distal renal tubular acidosis, is expressed in the red cell membrane but retained internally in kidney cells.

Human band 3 Walton is an AE1 mutation that results in the deletion of the 11 COOH-terminal amino acids of the protein and is associated with dominant distal renal tubular acidosis. The properties of band 3 Walton expressed with normal band 3 in the heterozygous mutant erythrocytes and the kidney isoform expressed in Xenopus oocytes and in the Madin-Darby canine kidney cell line were examined. The mutant erythrocytes have normal hematology but have reduced band 3 Walton content. Transport studies showed that erythrocyte band 3 Walton has normal sulfate transport activity, and kidney band 3 Walton has normal chloride transport activity when expressed in Xenopus oocytes. The mutant protein is clearly able to reach the cell surface of erythrocytes and oocytes. In contrast, while normal kidney band 3 was expressed at the cell surface in the kidney cell line, the Walton mutant protein was retained intracellularly within the kidney cells. The results demonstrate that band 3 Walton is targeted differently in erythrocytes and kidney cells and indicate that the COOH-terminal tail of band 3 is required to allow movement to the cell surface in kidney cells. It is proposed here that the mutant band 3 gives rise to dominant distal renal tubular acidosis by inhibiting the movement of normal band 3 to the cell surface. It is suggested that this results from the association of the normal and mutant proteins in band 3 hetero-oligomers, which causes the intracellular retention of normal band 3 with the mutant protein.

Band 3 mutations, distal renal tubular acidosis, and Southeast Asian ovalocytosis.

Familial distal renal tubular acidosis (dRTA) and Southeast Asian ovalocytosis (SAO) may coexist in the same patient. Both can originate in mutations of the anion-exchanger 1 gene (AE1), which codes for band 3, the bicarbonate/chloride exchanger in both the red cell membrane and the basolateral membrane of the collecting tubule alpha-intercalated cell. Dominant dRTA is usually due to a mutation of the AE1 gene, which does not alter red cell morphology. SAO is caused by an AE1 mutation that leads to a nine amino acid deletion of red cell band 3, but by itself does not cause dRTA. Recent gene studies have shown that AE1 mutations are responsible for autosomal recessive dRTA in several countries in Southeast Asia; these patients may be homozygous for the mutation or be compound heterozygotes of two different AE1 mutations, one of which is usually the SAO mutation.