Recessive Distal Renal Tubular Acidosis Research Articles

Molecular mechanisms of autosomal dominant and recessive distal renal tubular acidosis caused by SLC4A1 (AE1) mutations

Mutations of SLC4A1 (AE1) encoding the kidney anion (Cl(-)/HCO(3) (-)) exchanger 1 (kAE1 or band 3) can result in either autosomal dominant (AD) or autosomal recessive (AR) distal renal tubular acidosis (dRTA). The molecular mechanisms associated with SLC4A1 mutations resulting in these different modes of inheritance are now being unveiled using transfected cell systems. The dominant mutants kAE1 R589H, R901X and S613F, which have normal or insignificant changes in anion transport function, exhibit intracellular retention with endoplasmic reticulum (ER) localization in cultured non-polarized and polarized cells, while the dominant mutants kAE1 R901X and G609R are mis-targeted to apical membrane in addition to the basolateral membrane in cultured polarized cells. A dominant-negative effect is likely responsible for the dominant disease because heterodimers of kAE1 mutants and the wild-type protein are intracellularly retained. The recessive mutants kAE1 G701D and S773P however exhibit distinct trafficking defects. The kAE1 G701D mutant is retained in the Golgi apparatus, while the misfolded kAE1 S773P, which is impaired in ER exit and is degraded by proteosome, can only partially be delivered to the basolateral membrane of the polarized cells. In contrast to the dominant mutant kAE1, heterodimers of the recessive mutant kAE1 and wild-type kAE1 are able to traffic to the plasma membrane. The wild-type kAE1 thus exhibits a 'dominant-positive effect' relative to the recessive mutant kAE1 because it can rescue the mutant proteins from intracellular retention to be expressed at the cell surface. Consequently, homozygous or compound heterozygous recessive mutations are required for presentation of the disease phenotype. Future work using animal models of dRTA will provide additional insight into the pathophysiology of this disease.

Trafficking Defects of a Novel Autosomal Recessive Distal Renal Tubular Acidosis Mutant (S773P) of the Human Kidney Anion Exchanger (kAE1)

Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane.

Novel compound heterozygous SLC4A1 mutations in Thai patients with autosomal recessive distal renal tubular acidosis

Background: Mutations in the SLC4A1 gene have been found to cause either autosomal dominant (AD) or autosomal recessive (AR) distal renal tubular acidosis (dRTA). The SLC4A1 mutations causing AD dRTA were reported in white patients, whereas those associated with AR dRTA were often found in Southeast Asia. Here, the authors report additional novel SLC4A1 mutations in 3 patients with AR dRTA from 2 unrelated Thai families. Methods: The patients and members of their families were clinically studied. Red cell morphology and sulfate influx were examined. The SLC4A1 gene was screened, analyzed, and confirmed for mutations by molecular genetic techniques. Results: In the first family, the patient had dRTA, rickets, failure to thrive, nephrocalcinosis, and hypokalemic-hyperchloremic metabolic acidosis with a urine pH level of 7.00. He had novel compound heterozygous SLC4A1 G701D/S773P mutations, inherited from clinically normal heterozygous mother and father. In the second family, the patient and his sister had dRTA and Southeast Asian ovalocytosis (SAO) with different clinical severity. The patient had proximal muscle weakness, rickets, nephrocalcinosis, hypokalemia, normal anion gap metabolic acidosis, and urine pH level of 6.80. His sister was asymptomatic but the urine pH level could not be lowered to below 5.50 after a short acid load. Both siblings had compound heterozygous SLC4A1 SAO/R602H mutations. Conclusion: Two novel compound heterozygous SLC4A1 G701D/S773P and SAO/R602H mutations were identified in Thai patients with AR dRTA.

Anion exchanger 1 mutations associated with distal renal tubular acidosis in the Thai population.

We have previously demonstrated that compound heterozygous (SAO/G701D) and homozygous (G701D/G701D) mutations of the anion exchanger 1 (AE1) gene, encoding erythroid and kidney AE1 proteins, cause autosomal recessive distal renal tubular acidosis (AR dRTA) in Thai patients. It is thus of interest to examine the prevalence of these mutations in the Thai population. The SAO and G701D mutations were examined in 844 individuals from north, northeast, central, and south Thailand. Other reported mutations including R602H, DeltaV850, and A858D were also examined in some groups of subjects. The SAO mutation was common in the southern Thai population; its heterozygote frequency was 7/206 and estimated allele frequency 1.70%. However, this mutation was not observed in populations of three other regions of Thailand. In contrast, the G701D mutation was not found in the southern population but was observed in the northern, northeastern, and central populations, with heterozygote frequencies of 1/216, 3/205, and 1/217, and estimated allele frequencies of 0.23%, 0.73%, and 0.23%, respectively. The higher allele frequency of the G701D mutation in the northeastern Thai population corresponds to our previous finding that all Thai patients with AR dRTA attributable to homozygous G701D mutation originate from this population. This suggests that the G701D allele that is observed in this region might arise in northeastern Thailand. The presence of patients with compound heterozygous SAO/G701D in southern Thailand and Malaysia and their apparently absence in northeastern Thailand indicate that the G701D allele may have migrated to the southern peninsular region where SAO is common, resulting in pathogenic allelic interaction.

Mice lacking the B1 subunit of H +-ATPase have normal hearing

Acid–base homeostasis of endolymph is thought to be essential for normal inner ear function. This assumption was supported by clinical data from individuals affected by autosomal recessive distal renal tubular acidosis with sensorineural hearing loss. This recessive syndrome was recently demonstrated to be due to mutations in the gene encoding the B1 subunit of H +-ATPase ( ATP6B1). To examine the potential roles of H +-ATPase B1 subunit in inner ear development and function, we defined its spatial and temporal expression patterns in the developing mouse inner ear and examined the morphologic and physiologic effects of loss of its function. Standard in situ hybridization was used for the expression study with routine morphologic and physiologic assessments of hearing and balance in H +-ATPase B1 subunit ( Atp6b1) null mutant mice. Atp6b1 mRNA was first detected at embryonic day 11.5 (E11.5) in the endolymphatic duct epithelia. From E16.5 onward, Atp6b1 was also observed in the presumptive interdental cell layer of the spiral limbus in the cochlea. Auditory brainstem response tests revealed normal hearing in mice lacking Atp6b1. The inner ears of these mice develop normally and show no overt morphological abnormalities. Our data demonstrate that Atp6b1 is not critical for normal inner ear development or normal inner ear function in mice and suggest that other proton-transporting mechanisms or pH buffering systems must allow the mouse inner ear to compensate for lack of normal Atp6b1 activity.

ATP6B1 gene mutations associated with distal renal tubular acidosis and deafness in a child

A large proportion of autosomal recessive distal renal tubular acidosis (RTA) is associated with mutations in the ATP6B1 gene encoding the B1 subunit of H+-ATPase. H+-ATPase is one of the key membrane transporters for net acid excretion in the α-intercalated cells of the medullary collecting duct. Sensorineural hearing loss frequently accompanies this type of distal RTA. Mutational analysis of the ATP6B1 gene in a 9-year-old Korean boy with distal RTA and sensorineural hearing loss found 2 heterozygous missense point mutations. Although a single case report, this is the second report documenting ATP6B1 mutations in recessive distal RTA with sensorineural hearing loss after the original report by Karet et al and confirms the novelty of these mutations. Am J Kidney Dis 41:238-243. © 2003 by the National Kidney Foundation, Inc.

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of...

Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H +-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis

Several of the 13 subunits comprising mammalian H +-ATPases have multiple isoforms, encoded by separate genes and with differing tissue expression patterns, which may play an important role in the intracellular localization and activity of H +-ATPases. Here we report the cloning of three previously uncharacterized human genes, ATP6V1C2, ATP6V1G3 and ATP6V0D2, encoding novel H +-ATPase subunit isoforms C2, G3 and d2, respectively. We demonstrate that these novel genes are expressed in kidney and few other tissues, and confirm previous reports that the C1, G1 and d1 isoforms are ubiquitously expressed, while G2 is brain-specific. Previously we have shown that mutations in two kidney-specific genes, ATP6V1B1 and ATP6V0A4, encoding the H +-ATPase B1 and a4 subunit isoforms, cause recessive distal renal tubular acidosis (dRTA). As the genes reported here are expressed mainly in kidney, we assessed their candidacy as causative genes for recessive dRTA in eight kindreds unlinked to either known disease locus. Although no potential disease-causing mutations were seen in this cohort, this does not rule out a role for these genes in other families. The identification of these three novel tissue-specific isoforms supports the hypothesis that subunit differences may play a key role in the structure, site and function of H +-ATPases within the cell.

Autosomal recessive distal renal tubular acidosis caused by G701D mutation of anion exchanger 1 gene

Anion exchanger 1 (AE1 or band 3), encoded by the AE1 or SLC4A1 gene, regulates chloride-bicarbonate exchange in erythrocytes and α-intercalated cells of the distal nephron. Defects of AE1 at the basolateral membrane of α-intercalated cells may result in the failure of hydrogen ion secretion at the apical membrane, leading to distal renal tubular acidosis (dRTA). Abnormalities of the AE1 gene were previously reported to be associated with autosomal dominant dRTA. However, recent studies of Thai dRTA families have shown that mutations in this gene result in autosomal recessive (AR) dRTA, giving rise to the postulation that AE1 gene mutations causing AR dRTA might be found commonly in Thai pediatric patients with dRTA. We performed a study of the AE1 gene using DNA linkage, polymerase chain reaction single-strand conformation polymorphism, restriction endonuclease HpaII digestion, and DNA sequence analyses in eight families involving 12 Thai children with dRTA, shown by abnormal urinary acidification using a short acid-loading test, as well as among their family members. Seven patients with dRTA from five families had the same homozygous missense G701D mutation of the AE1 gene. Their parents or siblings heterozygous for the AE1 G701D mutation were clinically normal and did not have abnormal urinary acidification, although a heterozygous sibling in one family had abnormal urinary acidification. Results of this and previous studies show that a homozygous AE1 G701D mutation causes AR dRTA and is a common molecular defect among Thai pediatric patients with dRTA. © 2002 by the National Kidney Foundation, Inc.

Molecular cloning and characterization of Atp6n1b: a novel fourth murine vacuolar H+-ATPase a-subunit gene.

The 116-kDa a-subunit of the vacuolar proton pump (H(+)-ATPase) exists as several isoforms encoded by different genes and with different patterns of tissue expression. Its function within the multisubunit pump complex has yet to be elucidated. To date, three isoforms have been identified in mouse (designated a1-a3). We now report the cloning and characterization of Atp6n1b, encoding a novel fourth murine isoform (a4). Murine a4 has 833 residues and shows 85% amino acid identity to the human kidney-specific ATP6N1B protein in which loss-of-function alterations cause autosomal recessive distal renal tubular acidosis. The human and murine genes have similar genomic organization; furthermore, Atp6n1b maps to a region of mouse chromosome 6 that is syntenic with the segment of human 7q33-34 containing ATP6N1B. Together these findings establish the two genes as orthologs. The mouse a4 protein is 61, 52, and 47% identical to a1, a2, and a3, respectively. Phylogenetic analysis confirms that among vertebrates there are four a-subunit families, with a4 most resembling a1. Northern blot analysis of Atp6n1b reveals a 3.7-kilobase a4 transcript in kidney but not other major organs, and a reverse transcription polymerase chain reaction in 12 mouse tissues detects expression in kidney alone. Immunofluorescence studies in murine kidney demonstrate high intensity a4 staining at the surface of intercalated cells, with additional expression in the proximal tubule (not previously reported in human kidney). Similar apical a4 immunostaining is also present in male genital tissue. Identification of this novel murine kidney-enriched 116-kDa a-subunit provides a molecular tool for investigation of the currently unknown role of this protein, which is essential for proper function of the apical renal vacuolar H(+)-ATPase in man.

Unraveling the molecular basis of hereditary renal tubular acidosis

Renal tubular acidosis (RTA) is a clinical syndrome of disordered renal acidification in which the kidney fails to maintain a normal plasma concentration of HCO3 − in the setting of a normal rate of acid load from diet and metabolism. Most RTA in children is hereditary. Hereditary RTA can be classified into several groups on clinical and pathophysiological grounds. Recent progress in molecular biological analyses is unraveling the molecular basis of hereditary RTA. Mutations in the kidney type Na+/HCO3 − cotransporter gene (SLC4A4) cause permanent isolated proximal RTA with ocular abnormalities. Mutations in the carbonic anhydrase II gene lead to osteopetrosis, RTA (proximal RTA, distal RTA, or combined proximal and distal RTA), cerebral calcification, and mental retardation. Mutations in the gene (SLC4A1) encoding the Cl−/HCO3 − exchanger produce autosomal dominant distal RTA. However, mutations in SLC4A1 also link to a recessive syndrome of distal RTA with hemolytic anemia (Southeast Asian ovalocytosis) in Thailand. The phenotype of this distal RTA in Thailand is very close to that of autosomal dominant distal RTA in other countries. In contrast, autosomal recessive distal RTA, which is phenotypically heterogeneous, is a more severe disorder than autosomal dominant distal RTA. Mutations in ATP6B1, the B1-subunit of the apical H+-adenosine triphosphatase (ATPase) mediating distal nephron acid secretion, cause distal RTA with progressive bilateral sensorineural hearing loss. Moreover, mutations in ATP6N1B, encoding a tubular apical H+-ATPase 116-kD subunit, are reported to lead to distal dRTA with preserved hearing. These results can help in the further understanding of the molecular basis of hereditary RTAs and can help to characterize the clinical and genetic manifestations of these disorders.

Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing.

The multi-subunit H+-ATPase pump is present at particularly high density on the apical (luminal) surface of -intercalated cells of the cortical collecting duct of the distal nephron, where vectorial proton transport is required for urinary acidification. The complete subunit composition of the apical ATPase, however, has not been fully agreed upon. Functional failure of -intercalated cells results in a group of disorders, the distal renal tubular acidoses (dRTA), whose features include metabolic acidosis accompanied by disturbances of potassium balance, urinary calcium solubility, bone physiology and growth. Mutations in the gene encoding the B-subunit of the apical pump (ATP6B1) cause dRTA accompanied by deafness. We previously localized a gene for dRTA with preserved hearing to 7q33-34 (ref. 4). We report here the identification of this gene, ATP6N1B, which encodes an 840 amino acid novel kidney-specific isoform of ATP6N1A, the 116-kD non-catalytic accessory subunit of the proton pump. Northern-blot analysis demonstrated ATP6N1B expression in kidney but not other main organs. Immunofluorescence studies in human kidney cortex revealed that ATP6N1B localizes almost exclusively to the apical surface of -intercalated cells. We screened nine dRTA kindreds with normal audiometry that linked to the ATP6N1B locus, and identified different homozygous mutations in ATP6N1B in eight. These include nonsense, deletion and splice-site changes, all of which will truncate the protein. Our findings identify a new kidney-specific proton pump 116-kD accessory subunit that is highly expressed in proton-secreting cells in the distal nephron, and illustrate its essential role in normal vectorial acid transport into the urine by the kidney.

Localization of a Gene for Autosomal Recessive Distal Renal Tubular Acidosis with Normal Hearing ( rdRTA2) to 7q33-34

Failure of distal nephrons to excrete excess acid results in the "distal renal tubular acidoses" (dRTA). Early childhood features of autosomal recessive dRTA include severe metabolic acidosis with inappropriately alkaline urine, poor growth, rickets, and renal calcification. Progressive bilateral sensorineural hearing loss (SNHL) is evident in approximately one-third of patients. We have recently identified mutations in ATP6B1, encoding the B-subunit of the collecting-duct apical proton pump, as a cause of recessive dRTA with SNHL. We now report the results of genetic analysis of 13 kindreds with recessive dRTA and normal hearing. Analysis of linkage and molecular examination of ATP6B1 indicated that mutation in ATP6B1 rarely, if ever, accounts for this phenotype, prompting a genomewide linkage search for loci underlying this trait. The results strongly supported linkage with locus heterogeneity to a segment of 7q33-34, yielding a maximum multipoint LOD score of 8.84 with 68% of kindreds linked. The LOD-3 support interval defines a 14-cM region flanked by D7S500 and D7S688. That 4 of these 13 kindreds do not support linkage to rdRTA2 and ATP6B1 implies the existence of at least one additional dRTA locus. These findings establish that genes causing recessive dRTA with normal and impaired hearing are different, and they identify, at 7q33-34, a new locus, rdRTA2, for recessive dRTA with normal hearing.

Autosomal recessive distal renal tubular acidosis associated with Southeast Asian ovalocytosis

A defect in the anion exchanger 1 (AE1) of the basolateral membrane of type A intercalated cells in the renal collecting duct may result in a failure to maintain a cell-to-lumen H+ gradient, leading to distal renal tubular acidosis (dRTA). Thus, dRTA may occur in Southeast Asian ovalocytosis (SAO), a common AE1 gene abnormality observed in Southeast Asia and Melanesia. Our study investigated whether or not this renal acidification defect exists in individuals with SAO. Short and three-day NH4Cl loading tests were performed in 20 individuals with SAO and in two subjects, including their families, with both SAO and dRTA. Mutations of AE1 gene in individuals with SAO and members of the two families were also studied. Renal acidification in the 20 individuals with SAO and in the parents of the two families was normal. However, the two clinically affected individuals with SAO and dRTA had compound heterozygosity of 27 bp deletion in exon 11 and missense mutation G701D resulting from a CGG-->CAG substitution in exon 17 of the AE1 gene. Red cells of the two subjects with dRTA and SAO and the family members with SAO showed an approximate 40% reduction in sulfate influx with normal 4,4'-di-isothiocyanato-stilbene-2,2'-disulfonic acid sensitivity and pH dependence. These findings suggest that compound heterozygosity of abnormal AE1 genes causes autosomal recessive dRTA in SAO.