A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.     

Long-term adaptation of renal ion transporters to chronic diuretic treatment

Loop and thiazide diuretics are clinically useful to induce negative sodium balance. However, with chronic treatment, their effects tend to be blunted since the kidney adapts to diuretics. Molecular identification of the renal ion transporters has provided us with a new understanding of the mechanisms of intrarenal adaptation to diuretics at molecular levels. In the kidney, loop and thiazide diuretics are secreted from the proximal tubule via the organic anion transporter-1 (OAT1) and exert their diuretic action by binding to the Na-K-2Cl cotransporter type 2 (NKCC2) in the thick ascending limb and the Na-Cl cotransporter (NCC) in the distal convoluted tubule, respectively. Recent studies in animal models suggest that abundance of these ion transporters is affected by long-term diuretic administration. Downstream from the primary site of diuretic action, an increase in epithelial Na+ channel (ENaC) abundance is induced by chronic furosemide or hydrochlorothiazide treatment. This adaptation is consistent with previous reports showing cellular hypertrophy and increased Na+ absorption in distal tubular segments. The abundance of NKCC2 and NCC is increased by furosemide and hydrochlorothiazide, respectively. This compensatory upregulation suggests that either diuretic may activate the ion transporter within the primary site of action. In the proximal tubule, the abundance of OAT1 is increased by chronic treatment with furosemide or hydrochlorothiazide. This upregulation of OAT1 seems to be induced by substrate stimulation, lessening diuretic tolerance associated with long-term diuretic use.     

Identification of novel mutations in Na-Cl cotransporter gene in a Korean patient with atypical Gitelman's syndrome

The authors report the case of a 20-year-old man with unexplained hypokalemia and metabolic alkalosis suggesting hypokalemic tubulopathy. Interestingly, he showed a mixed phenotype of Gitelman's syndrome (GS) and Bartter's syndrome, which includes normomagnesemia, normal renal magnesium excretion, and hypocalciuria. Renal clearance study showed the presence of a critical defect in the distal nephron rather than loop of Henle. Further family study showed that his mother had a definitive phenotype of GS. By the molecular genetic analysis of these patients, 7 different mutations of the NCCT gene were identified consisting of 3 missense, 1 splice site, and 3 silent mutations. Four of these mutations were novel. The authors emphasize that the combination of a molecular genetic approach and renal clearance study could be of practical benefit in confusing clinical setting and support new diagnostic criteria in GS.     

Familial hypokalemia-hypomagnesemia or Gitelman's syndrome: a further case.

A woman aged 33 years presented hypokalemia and hypomagnesemia associated with renal potassium and magnesium wasting. Her mean 24-hour urinary calcium excretion was strikingly low despite normocalcemia, normal creatinine clearance, normal serum PTH and calcitriol. Normal distal fractional chloride reabsorption [CH2O/(CH2O + CCl)] was noted during water load but was reduced during hypotonic saline infusion. In response to intravenous furosemide (1 mg/kg), the patient showed significant increments in sodium, chloride and magnesium excretion as well as abolition of hypocalciuria. The association of renal calcium transport from magnesium transport together with exaggerated natriuresis after furosemide suggests the presence of a defect in the distal tubule rather than in the loop of Henle. We propose that our patient is affected by the syndrome of primary renotubular hypomagnesemia-hypokalemia with hypocalciuria, known as Gitelman's syndrome.     

Gitelman's syndrome (familial hypokalemia-hypomagnesemia)

Gitelman's syndrome (GS) is a heritable renal disorder characterized by hypomagnesemia, hypokalemia and hypocalciuria, and distinct from Bartter's syndrome (BS). As compared to those with BS, patients with GS present at an older age, and they have a milder clinical picture, normal or slightly decreased concentrating ability, reduced urinary excretion of calcium, and permanently decreased serum magnesium level. GS is caused by defective NaCl transport in the distal convoluted tubule, and linked to the gene encoding the thiazide sensitive Na-Cl-cotransporter located on chromosome 16q. Patients with BS, on the other hand, have mutations in the transporters in the thick ascending loop of Henle (NKCC2, ROMK, and C1C-Kb). Treatment of GS consists of magnesium salt replacement. Long term prognosis in terms of maintaining growth, preserving renal function and life expectancy is excellent.     

Hypokalemic alkalosis with hypocalciuria and normomagnesemia: A subgroup of Gitelman's syndrome?

We report a normomagnesemic patient with low normal blood pressure, hypokalemic alkalosis, hyperreninemia, hyperaldosteronism, and hypocalciuria. In renal clearance studies, distal delivery increased well and fractional distal solute reabsorption was dramatically diminished after the administration of furosemide, whereas thiazide produced no change in distal delivery and a moderate decrease in fractional distal solute reabsorption. These findings suggested that there may have been a defective locus in some part of the thiazide-sensitive segment of the distal convoluted tubule. As the features in this patient appear to be similar to those in Gitelman's syndrome, it is appropriate to designate this case, characterized by lack of hypomagnesemia, as a subgroup of Gitelman's syndrome. Received: February 12, 1998 / Accepted: June 30, 1998     

Novel mutations in thiazide-sensitive Na-Cl cotransporter gene of patients with Gitelman's syndrome

Gitelman's syndrome (GS) is an autosomal recessive disorder characterized by metabolic alkalosis, hypokalemia, hypomagnesemia, and hypocalciuria that has recently been reported to be linked to thiazide-sensitive Na-Cl cotransporter (TSC) gene mutations. In this study, possible mutations in the TSC gene of six Japanese patients clinically diagnosed with GS were investigated. Twenty-six exons encoding TSC were amplified by PCR and then completely sequenced by the direct sequencing method. Patient A showed a missense mutation of Arg 642 to Cys on the paternal allele and a missense mutation of Val 578 to Met and a 2-bp deletion (nucleotide 2543-2544) on the maternal allele. This deletion results in a frameshift that alters codon 837 to encode a stop signal rather than phenylalanine, and it is predicted to lead to loss of the latter half of the intracellular carboxy terminus. In the second family, two affected sisters, patients B and C, had a homozygous missense mutation of Thr 180 to Lys. Both of their parents, who are consanguineously married, have a heterozygous Thr180Lys mutation. Patient D has a homozygous mutation Thr180Lys, which is the same as the second family. Haplotype analysis indicates that patients B and C are not related to patient D. In patients E and F, we could identify only one mutant allele; Ala569Glu and Leu849His, respectively. All of the mutations identified are novel except for the Arg642Cys mutation, which has been found in a Japanese GS patient. Although further in vitro study is required to prove that the mutations are responsible for GS, it is possible that Thr180Lys and Arg642Cys mutations might be common mutations in Japanese GS.     

Studies on the mechanism of rubidium-induced kaliuresis

Renal clearance and electron microprobe methods were used 1) to elucidate the effects of chronic rubidium administration on potassium transport and 2) to localize, by the use of amiloride in acute experiments, the tubule site of interaction between rubidium and potassium. Substitution of drinking water by a 50 mM rubidium chloride solution for 9 to 11 days led to significant hypokalemia (plasma potassium 2.5 +/- 0.1 mM; plasma potassium plus rubidium 3.3 +/- 0.1 mM). Compared to a control group (reduction of plasma potassium to 3.4 +/- 0.1 mM by short-term potassium depletion) with a fractional potassium excretion of 2.1 +/- 0.3%, rubidium-treated rats excreted potassium at a much higher rate of 14.6 +/- 3.0%. The potassium content of principal cells was, however, significantly lower in rubidium-treated than in potassium-deprived animals. Similar to experiments in which rubidium was given acutely (3 hours), chronic rubidium administration was associated with preferential accumulation of rubidium in all tubule cells relative to potassium. Rubidium clearances were uniformly below those of potassium. Amiloride abolished the difference between rubidium and potassium clearances and sharply reduced the excretion of both cations. In view of the known site of action of amiloride, this suggests a distal tubule site of rubidium action on potassium transport. Amiloride also reduced or abolished the preferential uptake of rubidium into all but intercalated tubule cells. Marked cell heterogeneity of rubidium accumulation into intercalated cells was observed: One subpopulation, with low cell chloride, retained rubidium more effectively than another subpopulation with high cell chloride.     

Mimicry of surreptitious diuretic ingestion and the ability to make a genetic diagnosis

Gitelman's syndrome, also known as "hypocalciuric variant of Bartter's syndrome", is a cause of chronic hypokalemia and hypomagnesemia in adults. A specific gene has been found responsible for this disorder, encoding the thiazide-sensitive NaCl coporter (TSC) in the distal convoluted tubule. We describe a psychiatric patient with chronic symptomatic hypokalemia and hypomagnesemia whose electrolyte disturbances were subsequently misdiagnosed as an acute alcohol and benzodiazepine withdrawal syndrome, as chronic diuretic abuse and as a classical Bartter's syndrome. Finally, genetic investigation revealed the presence of mutations in the SLC12A3 gene leading to the proper diagnosis of Gitelman's syndrome. We emphasize that Gitelman's syndrome should be suspected in every hypokalemic patient with biochemical resemblance of diuretic ingestion, especially when repeated toxic screens for diuretics are negative. The ability to make a molecular-genetic diagnosis can be of practical benefit in confusing clinical settings.     

Linkage of Gitelman syndrome to the thiazide-sensitive sodium-chloride cotransporter gene with identification of mutations in Dutch families

Gitelman syndrome is a mostly autosomal recessive disorder affecting the renal tubular function associated with hypokalemia and hypomagnesemia. Functional studies point to a defect in the distal renal tubule in the thiazide-sensitive, electroneutral sodium-chloride cotransporter (TSC). Based upon the localization of a 2.6 cDNA encoding the human TSC to chromosome 16q13, polymorphic markers spanning the region from 16p12 to 16q21 were tested for linkage to the Gitelman syndrome locus in three Dutch families with autosomal recessive inheritance of this disorder. Using two-point linkage analysis, a maximum LOD score (Z_max of 4.49 (at Θ = 0.00) was found for the marker D16S408. One crucial recombination event places the Gitelman syndrome locus distal to D16S419 at 16q12-13. Subsequently we have tested our group of Gitelman patients for mutations in the human TSC gene. Two mutations were identified in three Gitelman families. Our study confirms that the human TSC gene is involved in Gitelman syndrome. Patients from three Gitelman families reveal two identical human TSC mutations, suggesting these families share a common ancestor. Received April 19, 1996; received in revised form and accepted May 24, 1996     

Distal nephron function in Bartter's syndrome: abnormal conductance to chloride in the cortical collecting tubule?

Five patients with the clinical patterns of Bartter's syndrome underwent a series of clearance studies in order to characterize the underlying tubule defect. Free water generation during maximal water diuresis (CH2O), expressed as percentage of the distal delivery (CH2O + CCl), was lower in the patients (72.5 +/- 3.2%) than in controls (84.4 +/- 5.5, p < 0.0001). During maximal water diuresis and furosemide administration (40 mg i.v. as bolus), NaCl reabsorption along the diluting nephron segments could be separated into 2 components, that occurring in the loop of Henle (DRNaHL) and that occurring in tubule segments beyond the macula densa (DRNaDT): DRNaHL was normal, while DRNaDT was reduced (3.1 +/- 0.8 vs. 6.2 +/- 2.5 ml/min in controls, p < 0.015). Thus, according to this furosemide protocol, our patients had normal solute reabsorption in the loop of Henle but reduced NaCl reabsorption in tubule segments beyond the macula densa. During 0.9% saline infusion (2 liters in 2 h, after stimulation of distal Na reabsorption with fludrocortisone) fractional excretion (FE) of K showed a linear rise with the increase of FECl-FEK, however, was much higher in the patients than in controls for every FECl level. In contrast, the infusion of Na2SO4, after fludrocortisone administration, induced similar FEK increases in patients and in controls. Thus, in these patients Na reabsorption in the distal nephron (possibly the cortical collecting tubule) was associated with the generation of a higher than normal electric potential gradient in the presence of Cl but not of another poorly reabsorbable anion, such as SO4(2-). These observations indicate that, in our patients, Henle's loop function is normal, while the collecting tubule function is abnormal. We suggest that NaCl wasting and enhanced tubular secretion of H+ and K in our patients might result from an abnormally low conductance to Cl in distal nephron site(s) where Na reabsorption is electrogenic, possibly the cortical collecting tubule. A larger than normal transtubular electric gradient would be generated by Na reabsorption, causing: (1) a direct stimulation of tubular secretion of K and H+ (leading to hypokalemia and alkalosis) and (2) inhibition of the reabsorption of Na ('trapped' into the tubular lumen by electric forces), with consequent extracellular volume contraction, hyperreninemia and hyperaldosteronism.     

Acquired Gitelman syndrome in a primary Sjögren syndrome patient with a SLC12A3 heterozygous mutation: A case report and literature review

Acquired Gitelman's syndrome (GS) associated with Sjögren syndrome (SS) is rare. A 50‐year‐old woman was admitted to our department because of nausea, acratia and sicca complex. Laboratory tests after admission showed renal failure, hypokalaemia, metabolic alkalosis, hypomagnesaemia and hypocalciuria, all of which met the diagnostic criteria for GS. Diagnostic evaluation identified primary SS as the cause of the acquired GS. Light microscopy of the renal tissue from the patient showed severe membranoproliferative glomerunephritis and tubulointerstitial nephritis. Immunohistochemical staining of the renal tissue showed the absence of sodium‐chloride co‐transporter (NCCT) in distal convoluted tubules. Genetic analysis of chromosomal DNA extracted from the patient's peripheral blood showed SLC12A3 gene heterozygous mutation. The reported case was comprehensively analyzed on the basis of the clinical features, and laboratory, pathological and genetic test findings. The patient has achieved a complete remission after meticulous care and appropriate treatment.     

An improved terminology and classification of Bartter-like syndromes

This Review outlines a terminology and classification of Bartter-like syndromes that is based on the underlying causes of these inherited salt-losing tubulopathies and is, therefore, more clinically relevant than the classical definition. Three major types of salt-losing tubulopathy can be defined: distal convoluted tubule dysfunction leading to hypokalemia (currently known as Gitelman or Bartter syndrome), the more-severe condition of polyuric loop dysfunction (often referred to as antenatal Bartter or hyperprostaglandin E syndrome), and the most-severe condition of combined loop and distal convoluted tubule dysfunction (antenatal Bartter or hyperprostaglandin E syndrome with sensorineural deafness). These three subtypes can each be further subdivided according to the identity of the defective ion transporter or channel: the sodium-chloride cotransporter NCCT or the chloride channel ClC-Kb in distal convoluted tubule dysfunction; the sodium-potassium-chloride cotransporter NKCC2 or the renal outer medullary potassium channel in loop dysfunction; and the chloride channels ClC-Ka and ClC-Kb or their beta-subunit Barttin in combined distal convoluted tubule and loop dysfunction. This new classification should help clinicians to better understand the pathophysiology of these syndromes and choose the most appropriate treatment for affected patients, while avoiding potentially harmful diagnostic and therapeutic approaches.     

Pathogenesis of hypokalemia in autosomal dominant hypocalcemia type 1

Background

Autosomal dominant hypocalcemia type 1 (ADH1) is a relatively rare endocrine disorder characterized by hypocalcemia and inadequate parathyroid hormone secretion. ADH is caused by activating mutations in the calcium-sensing receptor (CaSR) gene, CASR. CaSR plays a crucial role in calcium and magnesium homeostasis in the kidney. ADH may be accompanied by hypokalemia and metabolic alkalosis when it is classified as type V Bartter syndrome. However, the mechanism underlying hypokalemia in this disease is unclear.

Methods

We investigated a 33-year-old woman with hypocalcemia and hypoparathyroidism since childhood, whose mother also had hypocalcemia and hypoparathyroidism, but with no clinical symptoms. Blood examinations showed hypokalemia and metabolic alkalosis in the patient, but not her mother. We conducted mutation analysis and diuretic tests to clarify the patient’s and her mother’s diagnosis and to investigate the onset mechanism of hypokalemia in ADH1. We also determined the localization of CaSR in the kidney by immunohistochemistry.

Results

We detected a known gain-of-function mutation in CASR in both the patient and her mother. Diuretic tests revealed a response to furosemide and no reaction to thiazide in the patient, although the mother responded well to both diuretics. CaSR co-localized with the Na⁺–Cl^? cotransporter (NCCT) on distal tubular epithelial cells.

Conclusions

These results indicate that the NCCT in the distal convoluted tubule was secondarily affected in this patient. We conclude that the main pathogenesis of secondary hypokalemia in ADH1 in this patient was secondary NCCT dysfunction.

     

Bartter's syndrome due to a defect in salt reabsorption in the distal convoluted tubule

Bartter's syndrome is generally attributed to a primary defect in salt reabsorption either in the ascending limb of Henle's loop or in the proximal tubule. 2 siblings presented here have all the clinical and biochemical features of Bartter's syndrome but seem to have defective salt reabsorption in the distal convoluted tubule. A surreptitious use of diuretics was ruled out. Free water clearance was reduced in both patients and also was low after the addition of furosemide when compared with controls. Urine osmolalities following overnight dehydration were 883 and 1,000 mosm/l. The reduced maximal free water clearance argues against a proximal defect, and the normal urine concentration against a Henle's loop defect. Low free water clearance after furosemide suggests a defect in the distal convoluted tubule.     

A new mutation (intron 9 +1 G>T) in the SLC12A3 gene is linked to Gitelman syndrome in Gypsies

BACKGROUND: Gitel syndrome is an inherited tubular disorder characterized by metabolic alkalosis, hypokalemia, and hypomagnesemia of renal origin and hypocalciuria. The majority of patients with Gitelman syndrome carry inactivating mutations in the SLC12A3 gene encoding the sodium-chloride cotransporter located in the distal convoluted tubule. The purpose of this study was to investigate the underlying mutation in Gitelman syndrome patients of Gypsy race from different geographic origin. METHODS: Twenty Gypsy patients with clinical and biochemical features of Gitelman syndrome were investigated by mutational analysis. The patients belonged to 12 unrelated Gypsy families living in four different European countries. The parents and unaffected siblings of each patient, as well as the DNA of a population of 200 healthy control patients, were also analyzed. RESULTS: All patients were homozygous for the same splice site mutation, guanine to thymine in the first position of intron 9 of SLC12A3 gene. This mutation was not found in the control population. Parents were heterozygous for the mutation. Despite sharing a common mutation, the clinical manifestations of the syndrome in the patients varied from lack of symptoms in six children to severe growth retardation in four. CONCLUSION: Demonstration of a novel point mutation within the SLC12A3 gene in our cohort of Gypsy families with Gitelman syndrome is highly suggestive of a founder effect. This finding will facilitate the identification of the genetic defect in further cases of Gitelman syndrome among the Gypsy population. Our study represents the largest series ever published of patients with Gitelman syndrome having the same underlying mutation, and supports the lack of correlation between genotype and clinical phenotype in this disease.     

Identification of a novel R642C mutation in Na/Cl cotransporter with Gitelman's syndrome.

Gitelman's syndrome, a variant of Bartter's syndrome, is an inherited disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria, and these abnormalities have recently been linked to the thiazide-sensitive Na/Cl cotransporter (TSC) gene. We evaluated three unrelated patients affected with this syndrome whose diagnosis was made based on clinical and biochemical features. The data of clearance studies in these patients were compatible with Gitelman's syndrome. We then investigated possible mutations of the TSC gene. In one patient whose parents are consanguineous, we identified a novel missense mutation in the TSC gene, which causes alteration of arginine to cysteine at codon 642 (R642C mutation) located in the cytoplasmic tail of the product. This mutation results in the loss of an MspI site in exon 15 of the TSC gene. MspI digestion analysis of genomic DNA fragments from the family was consistent with the autosomal recessive inheritance of the disorder, and presence of this mutation correlated with the clinical manifestations. Such mutation was not detected in 47 normal healthy subjects. In the second patient, we found another missense mutation in one allele of the TSC gene, which results in alteration of arginine to glutamine at codon 955. In the third patient, no mutation causing amino acid substitution was found in the TSC gene. These results indicate that the R642C mutation in TSC is critically important for impairment of this cotransporter function and also suggest the necessity of further investigations in the genetic background of Gitelman's syndrome.     

“Chloride-shunt” syndrome: An overlooked cause of renal hypercalciuria

The case of a 7-year-old boy with the normotensive form of chloride-shunt syndrome is described. An unusual feature was the clinical presentation with lithiasis, caused by marked hypercalciuria of renal origin. The present studies were carried out to investigate the nature of the renal tubular defect. Indices for proximal and distal sodium chloride reabsorption were increased during hypotonic saline diuresis. Baseline sodium chloride excretion was low but increased above the range of control values after acute furosemide administration. Baseline potassium excretion was low, was not modified by the infusion of sodium chloride and increased significantly during infusions of sodium sulphate or sodium bicarbonate. Calcium excretion remained unchanged during sodium chloride, sodium sulphate or sodium bicarbonate infusions, but increased after furosemide administration. Nasal insufflation of 1-desamino-8-d-arginine-vasopressin induced both an increase in potassium excretion and a decrease in calcium and magnesium excretion. Plasma atrial natriuretic peptide was increased and was not significantly modified by infusion of hypertonic saline or acute administration of furosemide. These findings indicate that the primary renal abnormality appears to be an enhanced tubular reabsorption of sodium chloride, apparently present in the proximal tubule and the ascending loop of Henle. The associated presence of hypercalciuria also suggests a transport defect in the distal tubule. Decreased potassium excretion probably depends on a voltage-shunting defect in the cortical collecting tubule, which can be reversed by increasing the delivery of non-reabsobable anions or by enhancing the conductance of the luminal membrane. Treatment with hydrochlorothiazide was successful in correcting most biochemical abnormalities but did not result in catch-up growth.     

A novel mutation in the chloride channel gene,CLCNKB, as a cause of Gitelman and Bartter syndromes

BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary hypokalemic tubulopathies with distinct phenotypic features. GS has been considered a genetically homogeneous disorder caused by mutation in the gene encoding the NaCl cotransporter (TSC) of the distal convoluted tubule. In contrast, BS is caused by mutations in the genes encoding either the Na-K-2Cl cotransporter (NKCC2), the K+ channel (ROMK) or the Cl- channel (ClC-Kb) of the thick ascending limb. The purpose of this study was to examine the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel with hereditary hypokalemic tubulopathy. METHODS: Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent genetic linkage analysis. Mutation analysis was performed in affected individuals. RESULTS: Evaluation of affected family members (age range 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS included late age of presentation (>15 years) in 7 patients (58%), normal growth in 9 (75%), hypomagnesemia (SMg <0.7mmol/L) in 5 (42%), hypermagnesiuria (FEMg>5%) in 6 (50%) and hypocalciuria (urinary calcium/creatinine mmol/mmol <0.15) in 5 (42%). Features typical of CBS included early age of presentation (<1 year) in 3 (25%), polyuria/dehydration in 4 (33%), growth retardation in 3 (25%), hypercalciuria (urinary calcium/creatinine mmol/mmoverline>0.55) in 4 (33%) and nephrolithiasis in 1 (8%). Linkage analysis in affected patients excluded the TSC gene, SLC12A3, as the mutated gene, but demonstrated linkage to the Cl- channel gene, CLCNKB, on chromosome 1p36. Mutation analysis by direct sequencing revealed a novel homozygous missense mutation, arginine 438 to histidine (R438H), in exon 13 of CLCNKB in all patients. A restriction fragment length polymorphism (RFLP) analysis has been developed to aid in genotyping of family members. CONCLUSIONS: Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.     

Up-regulation of organic anion transporter 1 protein is induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney.

BACKGROUND: Thiazide and loop diuretics are secreted from the proximal tubule via the organic anion transport system to reach their principal sites of action. Recently, a multispecific organic anion transporter 1 (OAT1) was identified in rat kidney and was localized to the basolateral membrane of the S2 segment in the proximal tubule. We postulated that interactions between thiazide or loop diuretics and OAT1 may play a role in the adaptation to long-term diuretic use, and investigated whether OAT1 is regulated in vivo by chronic administration of diuretics at the protein level. METHODS: Semi-quantitative immunoblotting and immunohistochemistry were carried out in kidneys from male Sprague-Dawley rats using a polyclonal peptide-derived antibody to OAT1. Furosemide (12 mg/day/rat, n = 6), hydrochlorothiazide (3.75 mg/day/rat, n = 6) or vehicle (1.7% ethanolamine, n = 6) were infused subcutaneously for 7 days using osmotic minipumps. Experimental and vehicle-control rats were pair-fed, and two bottles of drinking water were provided, one containing tap water and the other containing a solution of 0.8% NaCl with 0.1% KCl. RESULTS: Overt diuretic responses were observed to both furosemide and hydrochlorothiazide infusions. There were no differences in body weight or creatinine clearance between the experimental and control rats. Although OAT1 protein abundance in cortical homogenates was increased by furosemide infusion (271 +/- 35 vs 100 +/- 15%, P < 0.05), Na-K-ATPase alpha1 subunit protein abundance was not affected (113 +/- 14 vs 100 +/- 8%, P = 0.42). Immunohistochemical localization in tissue sections confirmed a strong increase in OAT1 expression in the basolateral membrane of the S2 segment of proximal tubule. OAT1 protein abundance in cortical homogenates was also increased by hydrochlorothiazide infusion (181 +/- 25 vs 100 +/- 7%, P < 0.01), whereas Na-K-ATPase alpha1 subunit protein abundance was not affected (105 +/- 4 vs 100 +/- 4%, P = 0.34). CONCLUSION: Chronic furosemide or hydrochlorothiazide infusion caused increases in OAT1 protein abundance in rat kidney. These results suggest that OAT1 may be up-regulated in vivo by substrate stimulation at the protein level.