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.

Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I.

Pseudohypoaldosteronism type I (PHA1) is characterized by neonatal renal salt wasting with dehydration, hypotension, hyperkalaemia and metabolic acidosis, despite elevated aldosterone levels. Two forms of PHA1 exist. An autosomal recessive form features severe disease with manifestations persisting into adulthood. This form is caused by loss-of-function mutations in genes encoding subunits of the amiloride-sensitive epithelial sodium channel (ENaC; refs 2,3). Autosomal dominant or sporadic PHA1 is a milder disease that remits with age. Among six dominant and seven sporadic PHA1 kindreds, we have found no ENaC gene mutations, implicating mutations in other genes. As ENaC activity in the kidney is regulated by the steroid hormone aldosterone acting through the mineralocorticoid receptor, we have screened the mineralocorticoid receptor gene (MLR) for variants and have identified heterozygous mutations in one sporadic and four dominant kindreds. These include two frameshift mutations (one a de novo mutation), two premature termination codons and one splice donor mutation. These mutations segregate with PHA1 and are not found in unaffected subjects. These findings demonstrate that heterozygous MLR mutations cause PHA1, underscore the important role of mineralocorticoid receptor function in regulation of salt and blood pressure homeostasis in humans and motivate further study of this gene for a potential role in blood pressure variation.

Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III.

Analysis of patients with inherited hypokalaemic alkalosis resulting from salt-wasting has proved fertile ground for identification of essential elements of renal salt homeostasis and blood-pressure regulation. We now demonstrate linkage of this phenotype to a segment of chromosome 1 containing the gene encoding a renal chloride channel, CLCNKB. Examination of this gene reveals loss-of-function mutations that impair renal chloride reabsorption in the thick ascending limb of Henle's loop. Mutations in seventeen kindreds have been identified, and they include large deletions and nonsense and missense mutations. Some of the deletions are shown to have arisen by unequal crossing over between CLCNKB and the nearby related gene, CLCNKA. Patients who harbour CLCNKB mutations are characterized by hypokalaemic alkalosis with salt-wasting, low blood pressure, normal magnesium and hyper- or normocalciuria; they define a distinct subset of patients with Bartter's syndrome in whom nephrocalcinosis is absent. These findings demonstrate the critical role of CLCNKB in renal salt reabsorption and blood-pressure homeostasis, and demonstrate the potential role of specific CLCNKB antagonists as diuretic antihypertensive agents.

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.

Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion (Mg2+) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal Mg2+ wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into Mg2+ homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance.

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 rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

Increased bronchial responsiveness to histamine associated with shortened inhalation time.

A study was undertaken to assess the variability of results of histamine inhalation tests with the tidal breathing method, keeping the total dose of histamine delivered to the mouth constant but using either different nebulizers or the same Wright nebulizer operated under changing conditions. Seventeen asthmatic children had double histamine bronchial provocation tests with a Bennett-Twin nebulizer (output 0.31 ml/min, nebulization time 1 min) and a Wright nebulizer (output 0.15 ml/min, nebulization time 2 min), with a driving flow rate, in both instances, of 7 L/min. Another group of 15 asthmatic children had double provocation tests with a Wright nebulizer driven by a flow rate of either 7 L/min (output 0.15 ml/min, nebulization time 2 min) or 12 L/min (output 0.30 ml/min, nebulization time 1 min). In each experiment the volume of histamine solution delivered was about 0.30 ml. PC20-FEV1 was significantly lower, indicating an increased bronchial response, when both methods using "faster" delivery of histamine were used. We conclude that comparison of results obtained with histamine inhalation tests by the tidal breathing method cannot be made unless the nebulizer and operating criteria are the same.

[Long-term prognosis of childhood IgA nephropathy in adult life]. / Pronóstico en la vida adulta de la nefropatía IgA diagnosticada en la edad pediátrica.

BACKGROUND: To evaluate long-term prognosis in a group of children with IgA nephropathy and to analyse which clinical factors were associated with progression to chronic renal failure in adulthood. PATIENTS AND METHOD: Retrospective study. 58 young adults with IgA nephropathy diagnosed at 10.6(SD 2.9) years old and studied after a follow-up of 11.8 (SD 2.9) years. RESULTS: Relapses of macroscopic hematuria and proteinuria were the most frequent symptoms at onset (75.9%). In 25.9% of patients high plasmatic IgA levels were also detected. Most cases had grade I (44.8%)or grade II (44.8%) histological lesions at diagnosis. At the last control, clinical remision was observed in 21 patients (36.2%) and 50% of the whole group remained with abnormal urine. 8 patients(13.8%) reached terminal renal failure. Mean renal survival (defined as glomerular filtration rate above 50 ml/min/1.73 m2)was 100, 93.3 and 81.1% at 5, 10 and 15 years of evolution, respectively. CONCLUSIONS: About 14% of children with IgA nephropathy had long-term renal bad prognosis. Hypertension at onset, plasma creatinine elevation and proteinuria during adolescence were significant risk factors associated with chronic renal failure during adulthood. Minimal lesions at IgA nephropathy diagnosis in children did not exclude long-term poor prognosis.

Effect of docosahexaenoic acid administration on plasma lipid profile and metabolic parameters of children with methylmalonic acidaemia.

AIM: To evaluate the effect of administration of docosahexaenoic acid (DHA) on dyslipidaemia, plasma fatty acid composition and metabolic parameters of children with isolated methylmalonic acidaemia (MMA) (McKusick 25100). METHODS: Four children (3 male, 1 female) with MMA (mut(0)), participated in a crossover, randomized study of DHA administration (25 mg/kg per day, divided into three daily doses). The control group comprised 56 healthy children, aged 10+/- 2.7 years, (51 male, 5 female), who were followed in our clinic owing to possible familial risk of cardiovascular disease. RESULTS: The comparison of plasma fatty acid composition of children with MMA versus control children demonstrated that the patients had significantly higher values for oleic acid (p = 0.004) and linolenic acid (p = 0.008). No differences were observed in the levels of DHA and arachidonic acid. Plasma concentrations of insulin, glycine, ammonia, total cholesterol and cholesterol fractions did not change with DHA administration. No significant changes were observed in urinary excretion of methylmalonic acid. As expected, the percentage of DHA and n-3 fatty acids in plasma increased significantly after therapy (p = 0.005 and 0.014, respectively). The most remarkable result was a decrease of plasma levels of triglycerides after DHA therapy (p = 0.014). CONCLUSION: As previously found in normal children, dietary supplementation with DHA decreases the triglyceride levels, normalizing the hypertriglyceridaemia of these children without any evidence of short-term adverse effects.

Potassium homeostasis and its disturbances in children.

Although only 2% of the body potassium is present in the extracellular space, its concentration is finely regulated by the internal balance, or distribution of potassium between the intracellular and extracellular compartments, and by the external balance, or difference between intake and output of potassium. Internal balance is modulated by a host of factors, including insulin, epinephrine, extracellular pH and plasma tonicity. Potassium output from the body is mainly determined by renal excretion. Renal secretion of potassium takes place predominantly in the principal cells of late distal and cortical collecting tubules, by a process involving the accumulation of potassium in the cell by the activity of the basolateral Na+,K(+)-ATPase and its exit through luminal conductive channels. The factors regulating renal potassium secretion are potassium intake, rate of tubular fluid flow, distal sodium delivery, acid-base status and aldosterone. Hypokalaemia may result from a low potassium intake, excessive gastrointestinal, cutaneous or renal losses and altered body distribution. Aetiological diagnosis and therapy are best accomplished when the acid-base status is assessed at the same time. Before establishing the diagnosis of hyperkalaemia, spurious hyperkalaemia due to haemolysis or release of potassium from cells during clot retraction (pseudohyperkalaemia) should be ruled out. Hyperkalaemia may result from exogenous or endogenous loading, decreased renal output and altered body distribution. Acute hyperkalaemia represents an emergency situation which requires immediate therapy.

New insights into the pathogenesis of renal tubular acidosis--from functional to molecular studies.

The diagnosis and classification of renal tubular acidosis (RTA) have traditionally been made on the basis of functional studies. On these grounds, RTA has been separated into three main categories: (1) proximal RTA, or type 2; (2) distal RTA, or type 1; and (3) hyperkalemic RTA, or type 4. In recent years significant advances have been made in our understanding of the subcellular mechanisms involved in renal bicarbonate (HCO3-) and H+ transport. Application of molecular biology techniques has also opened a completely new perspective to the understanding of the pathophysiology of inherited cases of RTA. Mutations in the gene SLC4A4, encoding Na+-HCO3- cotransporter (NBC-1), have been found in proximal RTA with ocular abnormalities; in the gene SLC4A1, encoding Cl(-)-HCO3- exchanger (AE1), in autosomal dominant distal RTA; in the gene ATP6B1, encoding B1 subunit of H+-ATPase, in autosomal recessive distal RTA with sensorineural deafness; and in the gene CA2, encoding carbonic anhydrase II, in autosomal recessive osteopetrosis. Syndromes of aldosterone resistance have been also characterized molecularly and mutations in the gene MLR, encoding mineralocorticoid receptor, and in the genes SNCC1A, SNCC1B, and SCNN1G, encoding subunits of the epithelial Na+ channel, have been found in dominant and recessive forms of pseudohypoaldosteronism type 1, respectively. It can be concluded that, although functional studies are still necessary, a new molecular era in the understanding of disorders of renal acidification has arrived.

Bartter and related syndromes: the puzzle is almost solved.

It is now evident that the term Bartter syndrome does not represent a unique entity but encompasses a variety of disorders of renal electrolyte transport. Application of molecular biology techniques has permitted a better understanding of these "Bartter-like syndromes," which at present can be divided into three different genetic and clinical entities. Neonatal Bartter syndrome is observed in newborn infants and characterized by polyhydramnios, premature delivery, life-threatening episodes of fever and dehydration during the early weeks of life, growth retardation, hypercalciuria, and early-onset nephrocalcinosis. Two molecular defects have been identified: either at the gene encoding the renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) or the gene encoding an ATP-sensitive inwardly rectifying K channel (ROMK). "Classic" Bartter syndrome is mostly observed during infancy and childhood and is characterized clinically by polyuria and growth retardation. Nephrocalcinosis is not present. Very recently, either deletions or mutations at the gene encoding a renal chloride channel (ClC-Kb) have been identified. Gitelman syndrome is observed in older children and adults presenting with intermittent episodes of muscle weakness and tetany, hypokalemia, and hypomagnesemia. Mutations at the gene encoding the thiazide-sensitive Na-Cl cotransporter have been identified in the majority of patients studied. Obviously the validity of this classification must be confirmed in the near future when all mutations have been described and genotypic-phenotypic correlations are better defined.

Renal tubular hyperkalaemia in childhood.

Potassium output from the body is regulated by renal excretion, which takes place predominantly in the late distal and cortical collecting tubules. The accepted model for potassium secretion implies the accumulation of potassium into the cell by the activity of basolateral Na-K-ATPase and its exit through voltage-dependent conductive channels. The factors regulating renal potassium secretion are potassium intake, distal urinary flow, systemic acid-base equilibrium, aldosterone, antidiuretic hormone and, probably, epinephrine. Renal handling of potassium is best studied by the response to the acute administration of furosemide. This loop diuretic not only increases sodium and chloride excretion but also enhances potassium and hydrogen ion excretion and stimulates the renin-aldosterone axis. The term "renal tubular hyperkalaemia" refers to a tubular dysfunction where the hyperkalaemia is disproportionate to any reduction in glomerular filtration rate (GFR) and not due primarily or solely to aldosterone deficiency or to drugs impairing either mineralocorticoid action or tubular transport. The syndromes of renal tubular hyperkalaemia mainly observed in childhood are "chloride shunt" syndrome, hyporeninaemic hypoaldosteronism and primary or secondary pseudohypoaldosteronism. Differential diagnosis between these conditions is easily made if attention is paid to the level of GFR, presence of sodium wasting, activity of the renin-aldosterone axis and renal response to acute administration of furosemide.

Pathophysiology of the renal acidification defect present in the syndrome of familial hypomagnesaemia-hypercalciuria.

A distal acidification defect is frequently observed in the syndrome of familial hypomagnesaemia-hypercalciuria and hence this condition can be confused with primary distal renal tubular acidosis (RTA). This study demonstrates that in four unrelated patients with familial hypomagnesaemia-hypercalciuria the acidification defect is functionally different from that present in primary distal RTA. All patients exhibited hypomagnesaemia, hypermagnesuria, hypercalciuria, hyposthenuria, nephrocalcinosis and slight reduction of glomerular filtration rate (GFR). A moderate degree of metabolic acidosis was also present and basal data showed an inappropriately high urine pH (5.7-5.9) and a positive urine anion gap (Na + K-Cl = 11-28 mmol/l). Stimulation of distal acidification induced a fall in urine pH (4.7-5.6), but ammonium excretion remained low despite factoring by GFR (26-46 mumol/min per 1.73 m2, 35-54 mumol/100 ml GF). The urine to blood PCO2 gradient also remained low after sodium bicarbonate loading (1.3-17.7 mmHg). These results are best explained by both defective ammonia transfer to the deep nephron and impaired hydrogen ion secretion at the level of the medullary collecting duct, and probably are secondary effects of the medullary interstitial nephropathy.