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Biomedical subjects

F E Karet

Publications and source records attributed to F E Karet.

At least 19 recordsLinked to original sources
PubMed2000

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.

Acidosis, Renal Tubular↗
PubMed1999

Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness.

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.

Acidosis, Renal Tubular↗
PubMed1999

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.

Acidosis, Renal Tubular↗
PubMed1998

Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis.

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger AE1 have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in AE1. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in AE1 were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to AE1. In contrast, heterozygous mutations in AE1 were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a de novo mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in AE1 cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.

Acidosis, Renal Tubular↗
PubMed1997

Mutations contributing to human blood pressure variation.

In spite of a large body of physiological, biochemical, and recently genetic investigations, the causes of hypertension remain largely unknown. Recognition that hypertension is, in part, genetically determined has motivated studies to identify mutations conferring susceptibility. To date, mutations in at least 10 genes have been shown to alter blood pressure. The majority are rare mutations responsible for various mendelian hyper- and hypotensive syndromes, imparting large quantitative effects. Those causing hypertension are glucocorticoid-remediable aldosteronism, the syndrome of apparent mineralocorticoid excess, and Liddle's syndrome. Conversely, pseudohypoaldosteronism type 1, Bartter's, and Gitelman's syndromes all cause hypotension. In addition, mutations in the angiotensinogen gene are associated with hypertension. All these mutations alter blood pressure through a common pathway, affecting salt and water reabsorption in the kidney. These findings demonstrate the place of molecular genetic approaches in elucidating the underlying determinants of human blood pressure variation and may provide insight into the physiological mechanisms underlying common forms of hypertension.

Animals↗
PubMed1996

Localization of endothelin peptides in human kidney.

We investigated the synthesis and localization of endothelin isoforms in the human kidney using the reverse-transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. PCR products corresponding to the expected size for mRNA encoding ET-1, ET-2 and ET-3 were found in homogenates of renal medulla, cortex and vessels from each of five individuals. Using four rabbit polyclonal antibodies to assess the distribution of mature ET, Big ET-1, Big ET-2 and Big ET-3 immunoreactivity in the human kidney, mature IR ET localized to the cytoplasm of endothelial cells lining intra-renal blood vessels including interlobular and arcuate arteries, arterioles and adjacent arcuate veins, all of which showed strongly positive staining. IR Big ET-1 co-localized with the mature peptide. No specific staining was detected within these anatomical regions when pre-immune sera were substituted or primary antibody omitted. Mature IR ET also localized to the cytoplasm of endothelial cells within the glomerulus. Other capillary endothelial cells did not stain, and other structures stained only faintly by comparison. IR Big ET-2 and Big ET-3 could not be detected. These results show that human kidney contains mRNA encoding all three peptide isoforms, but only mature ET and Big ET-1 peptides could be detected by immunocytochemical staining. This provides further evidence that ET-1 may function as a renal peptide in humans, as it is locally synthesized within the kidney.

Base Sequence↗
PubMed1996

Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.

Maintenance of fluid and electrolyte homeostasis is critical for normal neuromuscular function. Bartter's syndrome is an autosomal recessive disease characterized by diverse abnormalities in electrolyte homeostasis including hypokalaemic metabolic alkalosis; Gitelman's syndrome represents the predominant subset of Bartter's patients having hypomagnesemia and hypocalciuria. We now demonstrate complete linkage of Gitelman's syndrome to the locus encoding the renal thiazide-sensitive Na-Cl cotransporter, and identify a wide variety of non-conservative mutations, consistent with loss of function alleles, in affected subjects. These findings demonstrate the molecular basis of Gitelman's syndrome. We speculate that these mutant alleles lead to reduced sodium chloride reabsorption in the more common heterozygotes, potentially protecting against development of hypertension.

Amino Acid Sequence↗
PubMed1996

Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2.

Inherited hypokalaemic alkalosis with low blood pressure can be divided into two groups-Gitelman's syndrome, featuring hypocalciuria, hypomagnesaemia and milder clinical manifestations, and Bartter's syndrome, featuring hypercalciuria and early presentation with severe volume depletion. Mutations in the renal Na-Cl cotransporter have been shown to cause Gitelman's syndrome. We demonstrate linkage of Bartter's syndrome to the renal Na-K-2Cl cotransporter gene NKCC2, and identify frameshift or non-conservative missense mutations for this gene that co-segregate with the disease. These findings demonstrate the molecular basis of Bartter's syndrome, provide the basis for molecular classification of patients with inherited hypokalaemic alkalosis, and suggest potential phenotypes in heterozygous carriers of NKCC2 mutations.

Amino Acid Sequence↗
PubMed1996

Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK.

Mutations in the Na-K-2Cl cotransporter (NKCC2), a mediator of renal salt reabsorption, cause Bartter's syndrome, featuring salt wasting, hypokalaemic alkalosis, hypercalciuria and low blood pressure. NKCC2 mutations can be excluded in some Bartter's kindreds, prompting examination of regulators of cotransporter activity. One regulator is believed to be ROMK, an ATP-sensitive K+ channel that 'recycles' reabsorbed K+ back to the tubule lumen. Examination of the ROMK gene reveals mutations that co-segregate with the disease and disrupt ROMK function in four Bartter's kindreds. Our findings establish the genetic heterogeneity of Bartter's syndrome, and demonstrate the physiologic role of ROMK in vivo.

Amino Acid Sequence↗
PubMed1996

Endothelin peptides and receptors in human kidney.

1. Animal kidneys are exquisitely sensitive to the vasoconstrictor and antinatriuretic effects of the endogenous vascular peptide endothelin. Animal studies have implicated endothelin in cyclosporin A and ischaemia-mediated renal damage. 2. In man, endothelin levels are raised in various disorders. Orally active endothelin antagonists are now being developed, but little was known of endothelin's role as a renal peptide in humans. These studies therefore aimed to characterize endothelin peptides and receptors ETA and ETB in human kidney, to direct potential therapeutic endeavours. 3. Ligand binding, immunocytochemical, radioimmunoassay and molecular biological studies were used to establish endothelin as a renal peptide in man. 4. The identification of species differences between man and rat directed further development of quantitative molecular biological methodology to permit analysis of endothelin receptors in human renal biopsies, and demonstrated perturbation of the system in the context of cyclosporin A therapy in renal transplantation.

Animals↗
PubMed1996

Selective downregulation of ETA receptor mRNA in renal transplant recipients on cyclosporin A revealed by quantitative RT-PCR.

Despite intensive investigation, a pathophysiological role for the endogenous vasoconstrictor peptide endothelin (ET) remains elusive. The kidney is particularly sensitive to the effects of ET, which are mimicked by the administration of cyclosporin A (CsA), and animal models suggest a role for ET in the vasoconstrictive effects of CsA. Using a recently validated novel fluorescent quantitative RT-PCR assay to enable the direct study of human renal biopsies, we have quantified mRNA for the two known ET receptor subtypes ETA and ETB in cortical tissue from three groups of patients: renal transplant recipients on CsA (n = 7), those with native renal disease (n = 5) and normal controls (n = 7). Median and mRNA levels (amol/microgram total RNA) were 0.024, 0.17 and 0.2 respectively for ETA and 0.57, 0.64 and 0.96 for ETB. These values indicate significant downregulation of ETA (P = 0.003) but not ETB (P = 0.104) mRNA in the transplant group. These results provide the first demonstration of a perturbation in the human ET system at tissue level in a pathophysiological situation, and suggest that the deleterious renal vasoconstrictor effects of CsA might be ameliorated by selective ETA receptor antagonism in the future. This study also illustrates the feasibility of ex vivo analysis of human diagnostic material at the molecular level.

Adult↗
PubMed1995

An endothelin-1 mediated autocrine growth loop involved in human renal tubular regeneration.

Renal tubules have the capacity to regenerate following injury. We have investigated the possibility that tubular-derived endothelins, acting as autocrine growth factors, may be involved in this response in human kidney. ET-1 immunoreactivity was demonstrated by immunohistochemical staining in proximal tubules, distal cortical tubules and medullary collecting ducts of human kidney. In cultured human renal proximal tubular cells, RNAase protection assays demonstrated the expression of ET-1 and ET-2 mRNA's, and radioimmunoassay, following separation of conditioned medium by reverse phase HPLC, showed immunoreactive material which co-eluted with ET-1 and ET-2. Competition binding studies revealed the presence of at least two types of endothelin receptor: one with high and one with low affinity for ET-3 relative to ET-1. Analysis of cellular RNA by RT-PCR demonstrated expression of mRNA's for both ETA and ETB receptor subtypes. Combined blockade of ETA and ETB receptors (by PD-145065) but not that of ETA receptors alone (by BQ-123) blocked the mitogenic effect of exogenous or endogenous ET-1 and also profoundly suppressed endogenous ET-1 synthesis. By contrast, incubation with the ETB receptor agonist, BQ-3020, stimulated endogenous ET-1 synthesis. Exposure of the cells to hypoxia (1% O2 for 16 to 24 hr) resulted in specific up-regulation of ET-1 but not ET-2 gene expression. These findings reveal the existence of a hypoxia-inducible, autocrine growth system in human proximal tubular cells, which is mediated by ET-1 through the ETB receptor, and which could function in vivo as an autoregenerative system for restoring tubular integrity after injury. The widespread distribution of ET-1 peptide in different tubular segment suggests that ET-1 mediated tubular regeneration may also occur in other nephron segments.

Binding, Competitive↗
PubMed1995

Comparative quantification of endothelin receptor mRNA in human kidney: new tools for direct investigation of human tissue.

By virtue of its exquisite sensitivity to the effects of endothelin-1 (ET-1), the kidney has been a consistent candidate for a pathophysiologic role for the endothelins. However, observed species differences in both receptor distribution and the subtypes mediating vasoconstriction have necessitated the development of a novel quantitative RT-PCR assay suitable for the direct investigation of human tissue, which is usually available only in very small amounts (i.e., biopsy specimens). In this study we quantified ETA and ETB receptor mRNA in normal renal cortex and medulla. For seven samples, cortex contained 0.19 +/- 0.10 amol ETA mRNA and 1.09 +/- 0.38 amol ETB mRNA/microgram total RNA (mean +/- SEM). In medulla these values were 0.47 +/- 0.25 and 2.17 +/- 1.90, respectively. The ratios of ETA to ETB were about 20:80, which correlates closely with previous studies of expressed receptor protein. This fluorescent nested quantitative RT-PCR assay provides a tool for further investigation of the human ET system at the molecular level in tissue from living individuals, and is of general applicability to the study of endogenous ligand-receptor systems.

Adult↗
PubMed1995

Characterization of peptide and nonpeptide antagonists in human kidney.

The human kidney contains about 70% endothelin ETB receptors, with the remaining ETA subtype mainly localized to the vasculature. Our aim was to characterize new ligands using native human receptors present in this tissue. In competition binding assays, sections of kidney (n > or = 3 individuals, +/- SEM) were incubated with 100 pM [125I]ET-1 and increasing concentrations of unlabeled ligands. The nonpeptide antagonists inhibited [125I]ET-1 binding monophasically (bosentan, Kd 360 +/- 50 nM, Bmax 39.5 +/- 9.4 fmol/mg protein; SB209670, Kd 80.0 +/- 12.5 nM, Bmax 51.8 +/- 20.4 fmol/mg protein). The ETB agonist sarafotoxin S6c competed biphasically with 1,400-fold selectivity for the ETB subtype (Kd ETA 2.2 +/- 0.2 microM, Bmax 22.6 +/- 4.9 fmol/mg protein; Kd ETB 1.5 +/- 0.2 nM, Bmax 46.3 +/- 9.0 fmol/mg protein). In contrast, BQ788 (an ETB antagonist in animals) competed monophasically (Kd 125.9 +/- 10.3 nM) and is not selective for the human renal ETB receptor. The ETA-selective antagonist S97-139 competed biphasically, with high affinity and 1,100-fold selectivity for the ETA site (Kd ETA 4.4 +/- 4.0 nM), but low affinity for ETB receptors (Kd ETB 5.1 +/- 0.4 microM). Autoradiography showed that ETA-selective compounds inhibited [125I]ET-1 binding to the ETA receptors mediating vasoconstriction in blood vessels but spared ETB receptors, which in the human kidney may be involved in salt and water balance as well as clearing ET from the plasma.

Autoradiography↗
PubMed1994

Bovril and moclobemide: a novel therapeutic strategy for central autonomic failure.

The consumption of tyramine-containing foods is contraindicated in patients on classic monoamine oxidase (MAO) inhibitors. We report successful therapeutic use of moclobemide (a MAO-A selective inhibitor) plus controlled amounts of Bovril (a tyramine-rich yeast-extract available as a food) in a patient with pure central autonomic failure who was rendered bed-bound by severe postural hypotension. Standing blood pressure is now at least 90/45 mm Hg. The selectivity of moclobemide allows about a tenth of ingested tyramine to reach nerve endings and thus the modest hypertensive effect of this combination re-established day-to-day function by restoring normotension.

Activities of Daily Living↗
PubMed1994

Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction.

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/micrograms total RNA. In medulla these values were 0.46 and 1.16 amol/micrograms, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens.

Base Sequence↗