Membrane Ion Transport in Bartter’s Syndrome
Evidence for a New Syndrome Subtype
Abstract Fifteen patients with Bartter’s syndrome (hyponatremic hypochloremic hypokalemic metabolic alkalosis) were compared with 15 healthy volunteers. Red blood cell Na+/H+ and Cl−/HCO3− exchanges were enhanced in all patients with Bartter’s syndrome. In calciuric normomagnesemic patients, sensitive to nonsteroidal anti-inflammatory drugs (classic Bartter’s syndrome), red blood cell Na+,K+,2Cl− cotransport was markedly reduced, calcium-dependent K+ permeability was moderately increased, and up to 60% of sodium permeability was represented by cAMP-activated fraction (presumably human analog of β-isoform of Na+/H+ exchange). In noncalciuric hypomagnesemic patients insensitive to indomethacin (Gitelman’s syndrome), Na+,K+,2Cl− cotransport was enhanced, Na+ permeability was increased due to calmodulin-dependent fraction, and calcium-dependent K+ permeability was markedly enhanced. A new subtype of Bartter-like syndrome (“variant Bartter’s syndrome”) has been described in which calciuria, hypomagnesemia, and insensitivity to nonsteroidal anti-inflammatory drugs were associated with decreased Na+,K+,2Cl− cotransport, enhanced calmodulin-activated fraction of Na+ influx, and reduced calcium-dependent K+ permeability.
- Na+-H+ exchanger
- Na+,K+,2Cl− cotransport
- Cl−/HCO3− exchange
- calcium-dependent potassium channels
- Bartter’s syndrome
In 1962 Bartter et al1 described renal tubular alkalosis associated with hyperreninemic hyperaldosteronism, elevated plasma concentrations of angiotensin II, normotension, and absent peripheral edema. The kidney biopsies of patients with Bartter’s syndrome showed juxtaglomerular hyperplasia, and elevated prostaglandin levels were found in their blood2 and urine.3 Some mechanisms have been proposed to explain this constellation of findings, such as resistance to the pressor action of angiotensin II,1 primary prostaglandin excess,4 and a defect in Na+ handling.5
Increased membrane permeability for sodium is a common finding in patients with Bartter’s syndrome, and is described in kidneys6 and extrarenal tissues, eg, erythrocytes.7 This defect, however, seems to be heterogeneous.8
While the classic Bartter’s syndrome is thought to be a serious disorder that impairs growth and leads to failure to thrive,9 its variant with hypocalciuria (“Gitelman’s syndrome”) appears to be a benign condition usually revealed in adulthood.9,10 Mutations of the renal Na+,K+,2Cl− cotransporter gene on chromosome 15q have been demonstrated in Bartter’s syndrome,11 and Gitelman’s syndrome has been attributed to mutations of the renal thiazide–sensitive Na+,Cl− cotransporter gene on chromosome 16.12 Whether the found alterations are ubiquitous for different cell types, is not known to date. It remains unproved that the revealed mutations cause really disturbed function of the exchangers they encode. It is also unclear whether the revealed alterations of the above-mentioned membrane transporters are the only ones seen in the Bartter-like syndromes. Since hyperaldosteronism may cause increased cell permeability for sodium per se,13 it is possible that at least part of the membrane alterations seen in Bartter-like syndromes can be attributed to the aldosterone effect rather than to the hereditary or genetic mechanisms.
This study has been carried out as a screening of several types of cation and anion transport systems in patients with Bartter-like syndromes. Red blood cells have been chosen for this evaluation as a convenient model with nearly absent genomic and rapid posttranscriptional changes in function and minimal interferences between the ion fluxes in the external and intracellular membranes.
Fifteen patients (9 women and 6 men) aged 16 to 36 years (mean±SEM, 28±4 years) with hypokalemic hyponatremic hypochloremic metabolic alkalosis, hyperreninemic hyperaldosteronism, normotension, and no peripheral edema were chosen as the study group. Fifteen volunteers (10 women and 5 men, aged 18 to 37 years, mean±SEM, 25±2 years) were enrolled into the control group after the results of routine blood tests had been obtained and determined to be normal. The characteristics of the groups are given in Table 1⇓. The laboratory tests and the membrane studies in both groups were carried out before treatment with spironolactone (200 mg/d for 7 days) and immediately thereafter.
Venous blood was sampled in plastic tubes containing heparin (20 to 50 IU/mL) after the patients had fasted for at least 4 hours (from 8 am to 1 pm) and kept ice-cold for no more than 12 hours.
Red Blood Cell Suspension
Red blood cells were sedimented at 1000g for 10 minutes and washed three times with a medium containing 150 mmol/L NaCl and 5 mmol/L bicarbonate-free phosphate buffer (1 mmol/L NaH2PO4: 5 mmol/L Na2HPO4, pH 7.4, 0°C to 4°C). The supernatant was removed together with the superficial cell layer, and red blood cells were used in the consecutive experiments.
Initial H+-Induced Na+ Permeability
In the iso-osmotic medium, the H+-dependent Na+ conductance is nearly quiescent.14 To induce this type of Na+ permeability, the cytoplasmic concentration of proton should be increased.15 For this purpose, 100 μL of the red blood cell suspension was preincubated, at 37°C, with 1.9 mL of a medium containing (in mmol/L) 150 NaCl, 1 KCl, 1 MgCl2, and 10 glucose. The pH was adjusted to 6.00 to 6.05 by the rapid addition of 0.2 mol/L HCl in 150 mmol/L choline-chloride. Anion exchanger was inhibited by 200 μmol/L DIDS. The pH was adjusted to 7.95 to 8.05 by the rapid addition of 0.05 mol/L NaOH. The kinetics of the proton efflux were registered by a pH meter. The H+ efflux was linear at least 3 seconds after a pHo of 8.00 had been achieved. The H+ permeability was calculated as ΔpHb/mt,where ΔpH is the initial rate of the medium acidification, b is the buffer capacity of the medium (determined by titration of 1.9 mL of the incubation medium with HCl and NaOH solutions, varied from 0.3 to 0.7 μmol H+/pH unit), m is the erythrocyte volume (taken as 100 μL), and t is the incubation time (5 minutes). The proton permeability under these conditions was considered equal to the H+-induced Na+-permeability.15,16
The evaluation of Na+ permeability was reassessed with 0.5 mmol/L amiloride added after DIDS. An amiloride-inhibited fraction of 0.5 mmol/L of the proton efflux was determined as Na+/H+ exchange,15 and its rate was calculated as (ΔpH−ΔpHamiloride)b/mt, where ΔpH is the initial rate of the medium acidification, in the absence and presence of amiloride, respectively.
The test of Na+ permeability was carried out without adding DIDS, but with the addition of 0.5 mmol/L amiloride before NaOH. NaOH was used with 1 mmol/L NaHCO3. Triton X-100 (0.2%) was used at the end of the Na+ permeability assessment to induce cell lysis, and the pH was registered as pHtriton. The experiment was performed with 200 μmol/L DIDS added before NaOH. Cl−/HCO3− exchange was determined as (pHtriton−pHDIDS/triton)b/mt. (For explanations of b, m, and t, see text above.)
Two hundred micromoles per liter of the red blood cell suspension was treated with 4 mCi/L 86Rb as a radioactive analogue of potassium. After 30 minutes of preincubation with a medium containing 150 mmol/L NaCl and 10 mmol/L Tris HCl (pH 7.4), the red blood cells were sedimented, washed twice with the same medium, and treated with 0.5 mL of 0.5% Triton X-100. After protein sedimentation, 1 mL of supernatant was transferred into Bray’s solution. The kinetics of the isotope influx was linear up to 75 minutes of incubation. The unidirectional ion flux was calculated as (A*−A)/amt, where A* and A are radioactivity of m mL red blood cells at 45 and 15 minutes of incubation (counts per minute [cpm]), a is the specific radioactivity of the medium (cpm/mmol), and t is the incubation time (in minutes).16 The experiment was reassessed with 250 μmol/L furosemide added to the medium before the isotope solution. Na+,K+,2Cl− cotransport was determined as furosemide-inhibited fraction of the 86Rb flux.
Calcium-Activated K+ Channels
Carbonylcyanide m-chlorophenyl hydrazone (10 mmol/L), a protonophore, was added to a medium containing (in mmol/L) 150 NaCl, 1 KCl, 1 MgCl2, and 10 glucose, at 37°C. Proton redistribution was initiated by the addition of A23187 (a calcium ionophore) and calcium (400 mmol/L CaCl2) and monitored by a pH meter. A23187 was thought to increase the cell calcium content and calcium-dependent K+ permeability and to cause leak of intracellular potassium into the medium, with subsequent hyperpolarization of the red blood cell membranes, erythrocyte uptake of protons from the medium, and alkalinization of the medium. The calcium-induced K+ permeability at 400 mmol/L was calculated as (pHcalcium/A23187−pHinitial)×RT/F, where R is the gas constant (2 cal/mol per °K), T is the absolute temperature (°K), and F is the Faraday’s constant (2.3×104 cal/V per mole).15
Na+ Permeability Modifiers
Calphostin C (50 nmol/L), KT 5720 (100 nmol/L), and W-13 (60 nmol/L) were used as inhibitors of PKC, cAMP-dependent kinase, and calmodulin-dependent kinase, respectively.
Concentrations of the modifiers were estimated in preliminary experiments (curves are not shown) and chosen as supramaximal.
Amiloride, DIDS, dimethyl sulfoxide, A23187, and furosemide were obtained from Sigma Chemical Co. Carbonylcyanide m-chlorophenyl hydrazone was obtained from Calbiochem Co. A 91-15 electrode was used connected to a PHM-64 (Radiometer) or a PBS-710 (El-Hama) pH meter.
The Kruskal-Wallis test (extension for small groups) was used to assess the differences between the groups. A value of P<.05 was considered statistically significant.
Na+/H+ and Cl−/HCO3− exchanges were enhanced in erythrocytes of all patients with Bartter-like syndrome (Table 2⇓) partly due to the PKC-dependent fraction of these fluxes.
In the classic Bartter’s syndrome (patients A through D), up to 60% of the NHE were paradoxically influenced by a cAMP-dependent kinase antagonist. Na+,K+,2Cl− cotransport values were significantly reduced in these patients, while the calcium-dependent K+ channels were only moderately stimulated.
The patients with Gitelman’s syndrome (patients E through J) exhibited enhanced amiloride-dependent NHE that was nonreactive to the inhibition of cAMP-dependent kinase, increased calmodulin-dependent fraction of NHE, and markedly enhanced calcium-dependent K+ permeability.
Five other patients (K through O) were calciuric (as in Bartter’s syndrome) and hypomagnesemic (as in Gitelman’s syndrome). They had a benign clinical course, and their elecrolyte disturbances did not respond to treatment with oral indomethacin (75 mg/d) (as in Gitelman’s syndrome). They had low Na+,K+,2Cl− cotransport, a negligible cAMP-dependent fraction and increased calmodulin-dependent fraction of NHE, and diminished calcium-dependent K+ efflux. These patients were thought to represent a “variant” Bartter’s syndrome.
Treatment with spironolactone (200 mg/d for 7 days) reduced NHE in all patients with Bartter-like syndrome (Table 2⇑) but failed to influence their laboratory parameters and activity of other membrane ion–transporting systems (data not shown).
Increased permeability of the cell membranes for Na+, usually measured in red blood cells, is a well-known phenomenon in Bartter’s syndrome.16-18 The degree of Na+ permeability differs between the patients with Bart-ter’s syndrome (Table 2⇑), as do Na+,K+,2Cl− antiport and calcium-dependent K+ permeability. Thus, a conclusion of heterogeneity of the Bartter’s syndrome (or Bartter-like syndromes) could be made. Indeed, even a preliminary analysis dissects Bartter’s syndrome into at least three different entities: (1) a type with hypercalciuria, normomagnesemia, increased cAMP-dependent NHE, nearly absent Na+,K+,2Cl− cotransport, increased calcium-activated K+ permeability, and a good effect of nonsteroidal anti-inflammatory drugs in the past (patients A through D, classic Bartter’s syndrome)1-4; (2) a type without calciuria, with hypomagnesemia, calmodulin-dependent enhancement of NHE, normal or increased Na+,K+,2Cl− cotransport, high calcium-dependent K+ permeability, and no effect of indomethacin (patients E through J, “Gitelman’s syndrome”)9,10; and (3) a presumably novel type of Bartter-like syndrome with hypercalciuria, hypomagnesemia, increased calmodulin-dependent NHE, diminished Na+,K+,2Cl− cotransport, very low calcium-dependent K+ efflux, and no effect of indomethacin (patients K through O, “variant” Bartter’s syndrome).
Decreased Na+,K+,2Cl− cotransport seems to be a cellular background for the classic Bartter’s syndrome. Impaired reabsorption of sodium, potassium, and chloride in the Henle’s loop explains perfectly its electrolyte disturbances. A mutation in the Na+,K+,2Cl− cotransporter gene NKCC2 on the long arm of chromosome 15 has been reported recently by Simon et al in families with Bartter’s syndrome.11 The same group has found a mutation in the ROMK gene encoding an ATP-sensitive K+ channel responsible for K+ recycling in the renal tubular cells that is supposed to be essential for normal activity of the Na+,K+,2Cl− cotransporter.19
In Gitelman’s syndrome, to the contrary, the Na+,K+,2Cl− exchanger acts regularly, and any additional membrane alteration, eg, decrease in the distal tube Na+,Cl− cotransport,20 can explain the similar changes. Indeed, mutations of the thiazide-sensitive Na+,Cl− cotransporter gene on chromosome 16 have been described in subjects with Gitelman’s syndrome.12 Markedly enhanced activity of maxi K+ channels (calcium-dependent K+ efflux), extrapolated to the apical membrane of the renal tubular cells, might explain renal potassium wasting21 in these patients, but the cause of overstimulation of the calcium-dependent K+ efflux in Gitelman’s syndrome remains obscure.
Reduced calcium-dependent K+ permeability in “variant” Bartter’s syndrome is a new and potentially interesting finding that could play a central role in the mechanism of electrolyte loss. Indeed, in the conditions of increased Na+/H+ exchange, cell swelling, upregulated stretch-dependent calcium channels and increased cytosolic calcium content, the basolateral potassium recycling is inhibited by increased intracellular calcium, and apical maxi K+ channels become an important route of K+ recycling necessary for normal functioning of the Na+,K+,2Cl− cotransporter.21 Thus, impaired calcium-dependent K+ permeability of the apical membranes could be a cause of inefficient Na+,K+,2Cl− transport in “variant” Bartter’s syndrome.
Salt depletion of the medulla apparently leads to the juxtaglomerular hyperplasia,1,4 hyperaldosteronism and increase in Na+ permeability. The enhancement of Na+/H+ exchange in the Bartter’s syndrome seems to be secondary to hyperaldosteronism1 since treatment with spironolactone reduces the NHE activity. Aldosterone increases the protein kinase C–dependent fraction of the Na+ fluxes (W. Koren, unpublished data), and its effect explains the elevated calphostin C–sensitive fractions of Na+ permeability in the Bartter-like syndromes (Table 2⇑).
Basolateral amiloride–sensitive NHE is generally found to be PKA/cAMP-independent.22 Contrary to this belief, the red blood cell NHE in the classic Bartter’s syndrome shows a significant fraction sensitive to PKA (when studied with a specific PKA inhibitor, KT 5720). This type of NHE also seems sensitive to PKC (indeed, inhibition of PKC also decreases the NHE activity, and the simultaneous inhibition of PKA and PKC leads to a more prominent decline in NHE activity than the inhibition of each kinase alone, Table 2⇑). Only one type of PKA-sensitive NHE (“β-NHE”) has been described in the trout red blood cells23 where it demonstrates independence of the intracellular pH and high sensitivity to β-adrenergic agonists. A PKA-sensitive type of human red blood cell NHE, which resembles the fish β-NHE, can be speculated to be coupled in the Bartter’s syndrome with the increased prostaglandin turnover that is generally found in these patients.4 This can also explain the unique effect of nonsteroidal anti-inflammatory drugs in this subset of patients. Vice versa, a lack of the cAMP-dependent fraction indirectly explains the insufficient effect of indomethacin in the two other Bartter-like syndromes. Enhanced cAMP-dependent activity in the classic Barrter’s syndrome may also explain the upregulation of the calcium-dependent K+ permeability, since the maxi K+ channels are cAMP/PKA-sensitive.21
The results of the present study confirm that patients with Bartter-like syndromes have widespread, if not ubiquitous, alterations in certain membrane cation and anion transport systems in cells of various types, some of which are secondary to hyperaldosteronism. At least some of these membrane alterations are present in subsets of patients with essential hypertension and hypertension due to primary aldosteronism.13,15 However, patients with Bartter-like syndromes remain normotensive. The mechanism responsible for blood pressure control in the Bartter’s syndrome still remains unknown.
Selected Abbreviations and Acronyms
|PKA||=||protein kinase A|
|PKC||=||protein kinase C|
The authors are grateful to Dr Hanan Gur and Professor Yehezkel Sidi for their essential assistance in this work.
Reprint requests to Wladimir Koren, MD, Institute for Hypertension, The Chaim Sheba Medical Center, Tel Hashomer 52621 Israel.
- Received February 12, 1997.
- Revision received March 12, 1997.
- Accepted June 20, 1997.
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