[Show abstract][Hide abstract] ABSTRACT: Final urinary acidification is achieved by electrogenic vacuolar H(+)-ATPases expressed in acid-secretory intercalated cells (ICs) in the connecting tubule (CNT) and the cortical (CCD) and initial medullary collecting duct (MCD), respectively. Electrogenic Na(+) reabsorption via epithelial Na(+) channels (ENaCs) in the apical membrane of the segment-specific CNT and collecting duct cells may promote H(+)-ATPases-mediated proton secretion by creating a more lumen-negative voltage. The exact localization where this supposed functional interaction takes place is unknown. We used several mouse models performing renal clearance experiments and assessed the furosemide-induced urinary acidification. Increasing Na(+) delivery to the CNT and CCD by blocking Na(+) reabsorption in the thick ascending limb with furosemide enhanced urinary acidification and net acid excretion. This effect of furosemide was abolished with amiloride or benzamil blocking ENaC action. In mice deficient for the IC-specific B1 subunit of the vacuolar H(+)-ATPase, furosemide led to only a small urinary acidification. In contrast, in mice with a kidney-specific inactivation of the alpha subunit of ENaC in the CCD and MCD, but not in the CNT, furosemide alone and in combination with hydrochlorothiazide induced normal urinary acidification. These results suggest that the B1 vacuolar H(+)-ATPase subunit is necessary for the furosemide-induced acute urinary acidification. Loss of ENaC channels in the CCD and MCD does not affect this acidification. Thus, functional expression of ENaC channels in the CNT is sufficient for furosemide-stimulated urinary acidification and identifies the CNT as a major segment in electrogenic urinary acidification.
Full-text · Article · Dec 2006 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: The identification of pathways that underlie common disease has been greatly impacted by the study of rare families that segregate single genes with large effect. Intracranial aneurysm is a common neurological problem; the rupture of these aneurysms constitutes a frequently catastrophic neurologic event. The pathogenesis of these aneurysms is largely unknown, although genetic and environmental factors are believed to play a role. Previous genomewide studies in affected relative pairs have suggested linkage to several loci, but underlying genes have not been identified. We have identified a large kindred that segregates nonsyndromic intracranial aneurysm as a dominant trait with high penetrance. Genomewide analysis of linkage was performed using a two-stage approach: an analysis of ~10,000 single-nucleotide polymorphisms in the 6 living affected subjects, followed by the genotyping of simple tandem repeats across resulting candidate intervals in all 23 kindred members. Analysis revealed significant linkage to a single locus, with a LOD score of 4.2 at 1p34.3-p36.13 under a dominant model with high penetrance. These findings identify a Mendelian form of intracranial aneurysm and map the location of the underlying disease locus.
Full-text · Article · Jan 2005 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jul 2003 · Hearing Research
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Dec 2001 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The relationship between salt homeostasis and blood pressure has remained difficult to establish from epidemiological studies of the general population. Recently, mendelian forms of hypertension have demonstrated that mutations that increase renal salt balance lead to higher blood pressure, suggesting that mutations that decrease the net salt balance might have the converse effect. Gitelman's syndrome, caused by loss of function mutations in the Na-Cl cotransporter of the distal convoluted tubule (NCCT), features inherited hypokalemic alkalosis with so-called "normal" blood pressure. We hypothesized that the mild salt wasting of Gitelman's syndrome results in reduced blood pressure and protection from hypertension. We have formally addressed this question through the study of 199 members of a large Amish kindred with Gitelman's syndrome. Through genetic testing, family members were identified as inheriting 0 (n=60), 1 (n=113), or 2 (n=26) mutations in NCCT, permitting an unbiased assessment of the clinical consequences of inheriting these mutations by comparison of the phenotypes of relatives with contrasting genotypes. The results demonstrate high penetrance of hypokalemic alkalosis, hypomagnesemia, and hypocalciuria in patients inheriting 2 mutant NCCT alleles. In addition, the NCCT genotype was a significant predictor of blood pressure, with homozygous mutant family members having significantly lower age- and gender-adjusted systolic and diastolic blood pressures than those of their wild-type relatives. Moreover, both homozygote and heterozygote subjects had significantly higher 24-hour urinary Na(+) than did wild-type subjects, reflecting a self-selected higher salt intake. Finally, heterozygous children, but not adults, had significantly lower blood pressures than those of the wild-type relatives. These findings provide formal demonstration that inherited mutations that impair renal salt handling lower blood pressure in humans.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2000 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause distal renal tubular acidosis, a condition characterized by impaired renal acid secretion resulting in metabolic acidosis. Patients with ATP6B1 mutations also have sensorineural hearing loss; consistent with this finding, we demonstrate expression of ATP6B1 in cochlea and endolymphatic sac. Our data, together with the known requirement for active proton secretion to maintain proper endolymph pH, implicate ATP6B1 in endolymph pH homeostasis and in normal auditory function. ATP6B1 is the first member of the H+-ATPase gene family in which mutations are shown to cause human disease.
[Show abstract][Hide abstract] ABSTRACT: Cerebral cavernous malformation (CCM) is frequently an inherited disorder showing autosomal dominant transmission. Genetic analysis has localized a gene causing CCM to a segment of the long arm of human chromosome 7 (7q). This evidence derives from investigation of a small number of families, mostly of Hispanic American descent. In this study, we have tested whether inherited CCM is always due to mutation in this 7q gene, or whether mutation in other genes can cause CCM.
We have studied subjects from two non-Hispanic families with inherited CCM. The clinical features of CCM in these families are indistinguishable from those in kindreds in which CCM is due to mutation in the 7q gene. To test whether CCM in these kindreds is caused by a mutation on 7q, we compared the inheritance of CCM to the inheritance of genetic markers on 7q.
Genetic analysis demonstrates independent inheritance of CCM and markers on 7q in both families studied. This evidence excludes mutation in the 7q gene as the cause of CCM in these families, with odds against CCM being due to mutation in 7q in each family of more than 100,000:1 and 100:1, respectively.
These findings demonstrate that inherited CCM is not always caused by a mutant gene on 7q, indicating the presence of at least a second gene in which mutation can cause CCM. These results have implications for genetic testing and the pathogenesis of this disorder.
[Show abstract][Hide abstract] ABSTRACT: Cerebral cavernous malformation is a vascular disease of the brain causing headaches, seizures, and cerebral hemorrhage. Familial and sporadic cases are recognized, and a gene causing familial disease has been mapped to chromosome 7. Hispanic Americans have a higher prevalence of cavernous malformation than do other ethnic groups, raising the possibility that affected persons in this population have inherited the same mutation from a common ancestor.
We compared the segregation of genetic markers and clinical cases of cavernous malformation in Hispanic-American kindreds with familial disease; we also compared the alleles for markers linked to cavernous malformation in patients with familial and sporadic cases.
All kindreds with familial disease showed linkage of cavernous malformation to a short segment of chromosome 7 (odds supporting linkage, 4X10(10).1). Forty-seven affected members of 14 kindreds shared identical alleles for up to 15 markers linked to the cavernous-malformation gene, demonstrating that they had inherited the same mutation from a common ancestor. Ten patients with sporadic cases also shared these same alleles, indicating that they too had inherited the same mutation. Thirty-three asymptomatic carriers of the disease gene were identified, demonstrating the variability and age dependence of the development of symptoms and explaining the appearance of apparently sporadic cases.
Virtually all cases of familial and sporadic cavernous malformation among Hispanic Americans of Mexican descent are due to the inheritance of the same mutation from a common ancestor.
Full-text · Article · May 1996 · New England Journal of Medicine