Thiazide-sensitive Sodium Chloride Cotransporter Research Articles

A case of Gitelman syndrome with severe hyponatraemia and hypophosphataemia

Gitelman syndrome (GS) is a renal tubular disorder of the thiazide-sensitive sodium chloride cotransporter, which is located in the distal tubule of the loop of Henle. We present a rare case of GS complicated by severe hyponatraemia and hypophosphataemia. A 17-year-old boy was admitted to our institution with fever and lethargy. The workup revealed typical features of GS, i.e. hypokalaemia, hypomagnesaemia and metabolic alkalosis. In this report, we discuss the differential diagnoses and rationale for accepting GS as the most likely diagnosis. This case was complicated by severe hyponatraemia (115 mmol/L) and hypophosphataemia (0.32 mmol/L). We concluded that the syndrome of inappropriate secretion of antidiuretic hormones could not be ruled out and that respiratory alkalosis was the most likely aetiology of hypophosphataemia. This case report also generates an interesting discussion on water and electrolyte metabolism.

Gitelman syndrome combined with complete growth hormone deficiency.

Gitelman syndrome is a rare autosomal recessive hereditary salt-losing tubulopathy, that manifests as hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. It is caused by mutations in the solute carrier family 12(sodium/chloride transporters), member 3 (SLC12A3) gene encoding the thiazide-sensitive sodium chloride cotransporter channel (NCCT) in the distal convoluted tubule of the kidney. It is associated with muscle weakness, cramps, tetany, vomiting, diarrhea, abdominal pain, and growth retardation. The incidence of growth retardation, the exact cause of which is unknown, is lower than that of Bartter syndrome. Herein, we discuss the case of an overweight 12.9-year-old girl of short stature presenting with hypokalemic metabolic alkalosis. The patient, on the basis of detection of a heterozygous mutation in the SLC12A3 gene and poor growth hormone (GH) responses in two provocative tests, was diagnosed with Gitelman syndrome combined with complete GH deficiency. GH treatment accompanied by magnesium oxide and potassium replacement was associated with a good clinical response.

Renal phosphate handling in Gitelman syndrome—the results of a case–control study

Patients with Gitelman syndrome, a hereditary salt-wasting tubulopathy, have loss-of-function mutations in the SLC12A3 gene coding for the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule. Since the bulk of filtered phosphate is reabsorbed in the proximal tubule, renal phosphate wasting is considered exceptional in Gitelman syndrome. We investigated the renal handling of inorganic phosphate in 12 unselected Italian patients affected with Gitelman syndrome (5 females and 7 males, aged 6.0-18 years, median age 12 years) and in 12 healthy subjects matched for gender and age (controls). The diagnosis of Gitelman syndrome among the patients had been made clinically and confirmed by molecular biology studies. The biochemical hallmarks of Gitelman syndrome, namely hypochloremia, hypokalemia, hypomagnesemia, increased urinary excretion of sodium, chloride, potassium and magnesium and reduced urinary excretion of calcium, were present in the 12 patients. In addition, both the plasma inorganic phosphate concentration (median and interquartile range: 1.28 [1.12-1.36] vs. 1.61 [1.51-1.66)] mmol/L) and the maximal tubular reabsorption of inorganic phosphate (1.08 [0.99-1.22] vs. 1.41 [1.38-1.47] mmol/L) were significantly lower (P < 0.001) in Gitelman patients than in control subjects. Circulating levels of 25-hydroxyvitamin D, intact parathyroid hormone and osteocalcin were similar in patients and controls. The results of our case-control study disclose a hitherto unrecognized tendency towards renal phosphate wasting with mild to moderate hypophosphatemia in Gitelman syndrome.

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/-) mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.

Activation of Thiazide-Sensitive Co-Transport by Angiotensin II in the cyp1a1-Ren2 Hypertensive Rat

Transgenic rats with inducible expression of the mouse Ren2 gene were used to elucidate mechanisms leading to the development of hypertension and renal injury. Ren2 transgene activation was induced by administration of a naturally occurring aryl hydrocarbon, indole-3-carbinol (100 mg/kg/day by gastric gavage). Blood pressure and renal parameters were recorded in both conscious and anesthetized (butabarbital sodium; 120 mg/kg IP) rats at selected time-points during the development of hypertension. Hypertension was evident by the second day of treatment, being preceded by reduced renal sodium excretion due to activation of the thiazide-sensitive sodium-chloride co-transporter. Renal injury was evident after the first day of transgene induction, being initially limited to the pre-glomerular vasculature. Mircoalbuminuria and tubuloinsterstitial injury developed once hypertension was established. Chronic treatment with either hydrochlorothiazide or an AT1 receptor antagonist normalized sodium reabsorption, significantly blunted hypertension and prevented renal injury. Urinary aldosterone excretion was increased ∼20 fold, but chronic mineralocorticoid receptor antagonism with spironolactone neither restored natriuretic capacity nor prevented hypertension. Spironolactone nevertheless ameliorated vascular damage and prevented albuminuria. This study finds activation of sodium-chloride co-transport to be a key mechanism in angiotensin II-dependent hypertension. Furthermore, renal vascular injury in this setting reflects both barotrauma and pressure-independent pathways associated with direct detrimental effects of angiotensin II and aldosterone.

SPAK, OSR1 and Cab39/MO25 form an Interdependent Signaling system which Regulates Thiazide‐Sensitive Salt‐transport, Distal Tubule Mass and Blood Pressure

STE20/SPS‐1‐related proline‐alanine‐rich protein kinase (SPAK) and, Oxidative Stress Related Kinase (OSR1), co‐localize at the apical membrane of the Thick ascending Limb (TAL) and Distal Convoluted Tubule (DCT) and both regulate the potassium‐dependent sodium‐chloride co‐transporter, NKCC2, thiazide‐sensitive sodium‐chloride cotransporter, NCC in vitro. Yet genetic ablation of SPAK in mice causes a salt‐wasting nephropathy that is restricted to the DCT, reminiscent of Gitelman's syndrome. Here, we explore why proper DCT function is especially SPAK‐dependent. In the TAL of SPAK−/− mice, OSR1 and Cab39/MO25, a newly described OSR1/SPAK regulatory protein, remain at the apical membrane where they function with a compensatory increase in the AMP‐activated kinase (AMPK) to hyper‐phosphorylate NKCC2. By contrast, the OSR1/SPAK/M025 signal transduction apparatus is completely disrupted in the DCT. OSR1 and MO25 become largely displaced from the thiazide‐sensitive sodium‐chloride cotransporter, NCC, and the apical membrane. OSR1 redistributes to dense punctate structures within the cytoplasm. These changes are paralleled by a dramatic decrease in NCC abundance and phosphorylation. Without SPAK and the proper localization of OSR1 and MO25, phosphorylation‐dependent regulation of NCC by dietary sodium restriction is lost. SPAK−/− mice also exhibit a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the interdependent nature of OSR1 and MO25 on SPAK in the DCT, SPAK−/− mice are highly sensitive to dietary salt‐restriction, displaying prolonged negative sodium balance and hypotension.

Localization of Tubular Adaptation to Renal Sodium Loss in Gitelman Syndrome

Gitelman syndrome (GS) is a salt-wasting tubulopathy that results from the inactivation of the human thiazide-sensitive sodium chloride cotransporter located in the distal convoluted tubule. Tubular adaptation to renal sodium loss has been described and localized in the distal tubule in experimental models of GS but not in humans with GS. The tubular adaptation to renal sodium loss is described. Osmole-free water clearance and endogenous lithium clearance with furosemide infusion are used to compare 7 patients with genetically confirmed GS and 13 control participants. Neither endogenous lithium clearance nor osmole-free water clearance disclosed enhanced proximal fluid reabsorption in patients with GS. These patients displayed significantly lower osmole-free water clearance factored by inulin clearance (7.1 ± 1.9 versus 10.1 ± 2.2; P<0.01) and significantly lower fractional sodium reabsorption in the diluting nephron (73.2% ± 7.1% versus 86.1% ± 4.7%; P<0.005), consistent with the inactivation of the thiazide-sensitive sodium chloride cotransporter. The furosemide-induced reduction rate of fractional sodium reabsorption in the diluting segment was higher in patients with GS (75.6% ± 6.1% versus 69.9% ± 3.2%; P<0.039), suggesting that sodium reabsorption would be enhanced in the cortical part of the thick ascending limb of the loop of Henle in patients with GS. These findings suggest that tubular adaptation to renal sodium loss in GS would be devoted to the cortical part of the thick ascending limb of the loop of Henle in humans.

Pathogenesis of calcineurin inhibitor–induced hypertension

This article reviews the current understanding of the mechanisms of calcineurin inhibitor-induced hypertension. Already early after the introduction of cyclosporine in the 1980s, vasoconstriction, sympathetic excitation and sodium retention by the kidney had been shown to play a role in this form of hypertension. The vasoconstrictive effects of calcineurin inhibitors are related to interference with the balance of vasoactive substances, including endothelin and nitric oxide. Until recently, the renal site of the sodium-retaining effect of calcineurin inhibitors was unknown. We and others have shown that calcineurin inhibitors increase the activity of the thiazide-sensitive sodium chloride cotransporter through an effect on the kinases WNK and SPAK. Here, we review the pertinent literature on the hypertensinogenic effects of calcineurin inhibitors, including neural, vascular and renal effects, and we propose an integrated model of calcineurin inhibitor-induced hypertension.

Gitelman′s syndrome with panhypopituitarism: Reno-endocrine interplay

Gitelman's syndrome is an inherited tubulopathy affecting thiazide-sensitive sodium chloride cotransporter, which manifests with hypokalemic alkalosis, hypomagnesemia, and hypocalciuria. Recently few cases have been described having an association of Gitelman's syndrome with pituitary abnormalities on imaging, though with normal hormonal status. We describe the first case of an adult patient having Gitelman's syndrome and hypopituitarism with abnormal pituitary imaging. She presented to us with hypotension, hypokalemia, hypomagnesemia with alkalosis, hypothyroidism, hypocortisolism, and hypogonadism. She was treated with replacement of electrolytes and hormones, to which she showed an excellent response.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1

The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+-sensitive K+-selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium–chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+/K+-ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit.Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo.Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo.

Identification of a Novel WNK4 Mutation in Chinese Patients with Pseudohypoaldosteronism Type II

Background: It has been reported that mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type II (PHA2), an autosomal dominant renal disease. WNK kinase proteins are expressed in the kidney and regulate ion transport including the thiazide-sensitive sodium chloride cotransporter (NCC). In this report, we screened 4 Chinese PHA2 pedigrees for WNK4 mutations, identified a novel mutation, and studied its effects on NCC protein trafficking in vitro. Methods: The patients’ genomic DNA was extracted from peripheral leukocytes. Sequence analysis was performed by PCR amplification of the 19 exons of WNK4. The wild-type or mutant WNK4 was coexpressed with NCC in HEK293 cells. We measured the effect of wild-type WNK4 compared to the mutant WNK4 on NCC protein surface expression. Results: A novel missense mutation in WNK4, K1169E, was identified in 1 of the 4 pedigrees. Analysis of confocal images showed that K1169E lost its inhibitory effect on NCC surface expression compared to wild-type WNK4 when expressed in HEK293 cells, while it did not change NCC total protein expression. Conclusions: We identified an unreported disease-causing WNK4 missense mutation, K1169E, in 1 Chinese PHA2 pedigree. This mutation appears to be a ‘loss of function’ of NCC inhibition and suggests that some important functional roles lie in the 2nd coiled-coil domain of WNK4.

Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) to assess the role of arginine vasopressin (AVP) in regulating NCC in rodent kidney in vivo. Immunohistochemistry showed distinct staining of phosphorylated NCC (pNCC) at the apical plasma membrane domain of distal convoluted tubule (DCT) cells. Unlike total NCC, pNCC was localized only to the apical plasma membrane as determined by immunogold electron microscopy. In AVP-deficient Brattleboro rats, acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) exposure significantly increased pNCC abundance at the apical plasma membrane by about threefold, whereas total NCC and its cellular distribution were not affected. dDAVP significantly increased the abundance of phosphorylated STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinase (SPAK and OSR1), kinases implicated in NCC phosphorylation. Intracellular calcium levels in early and late DCTs were increased in response to 1 min superfusion of dDAVP, confirming that these segments are AVP responsive. In rats fed a high-salt diet with angiotensin (ANG) type 1-receptor blockade, similar increases in pNCC and active SPAK and OSR1 were detected following chronic or acute dDAVP, thus indicating the effects of AVP are independent of ANGII. Our results show that AVP is a potent regulator of NCC activity.

Hypokalemic rhabdomyolysis in a child with Gitelman’s syndrome

We report here the first published case of a pediatric patient with Gitelman's syndrome (GS) in whom hypokalemia-associated rhabdomyolysis developed. A 13-year-old girl was admitted with weakness of the extremities, walking difficulty and calf pain. Laboratory data showed a serum potassium level of 2.1 mmol/l and a serum creatinine phosphokinase level of 1,248 IU/l plus myoglobinemia. The presence of normomagnesemia was the basis for a genetic analysis of the thiazide-sensitive sodium chloride cotransporter gene, which revealed compound heterozygous mutations in this gene. Prompt fluid expansion and potassium supplementation led to regression of the muscle symptoms. Hypokalemia can be a rare cause of rhabdomyolysis in patients with GS, even in childhood. We emphasize that genetic analysis is advisable to determine whether the suspicion of GS is warranted.

Rare but Relevant Kidney Disorders

Occurrence of hypomagnesemia (serum Mg2+ levels <0.7 mmol/L) in the general population has been estimated to be approximately 2%. The kidney is central for the maintenance of the Mg+ balance. A large body of knowledge has been generated from the study of inherited defects of renal Mg2+ handling. The majority of filtered Mg+ is reabsorbed along the proximal tubule and thick ascending limb of Henle's loop via a passive paracellular pathway. Claudin 16, formerly paracellin 1, and claudin 19 are renal tight junction proteins that are important for paracellular reabsorption of calcium and magnesium in thick ascending limb. Mutations of the corresponding genes, CLDN 16 and 19, result in a rare autosomal recessive tubular disorder that is characterized by familial hypomagnesemia, hypercalciuria, and nephrocalcinosis (FHHNC). This disease is frequently associated with progressive renal failure as a result of both intrarenal calcium deposition and additional roles of claudins in the maintenance of tight junction integrity. Children with a complete loss of function of CLDN16 have a more rapid progression of renal failure than those with residual function (1). The thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule (DCT) is mutated in Gitelman syndrome. Hypokalemic metabolic alkalosis and hypocalciuria are associated with hypomagnesemia. At the apical membrane of DCT cells is also present the Mg2+-permeable transient receptor potential cation channel, subfamily M (TRPM6). Mg2+ uptake from the tubular fluid via TRPM6 is primarily driven by the negative potential across the luminal membrane. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6. Mutations in FXYD2, encoding the γ-subunit of the basolateral Na+-K+-ATPase, is responsible for dominant isolated renal Mg2+ wasting, as a result of misrouting of this subunit. This defect has been described so far in a single family (but read this summary to the end). Finally, the recently discovered magnesiotropic hormone EGF causes isolated recessive renal hypomagnesemia (see references in Glaudemans et al.). Glaudemans et al. identified a new mechanism for autosomal dominant hypomagnesemia. A large Brazilian family was studied. Twenty-one affected members had serum Mg2+ levels <0.4 mmol/L, which was responsible for muscle symptoms and tetany from infancy. The locus was identified on chromosome 12. The mutation was found in KCNA1, a gene encoding the voltage-gated K+ channel, Kv1.1. This channel co-localized with TRPM6 along the luminal membrane of DCT. Upon overexpression in a human kidney cell line, patch-clamp analysis revealed that the mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. The negative potential across the apical membrane is normally maintained by an apical K+ efflux via Kv1.1 energized by the action of Na+-K+-ATPase. When Kv1.1 function is lost, Mg2+ reabsorption in DCT is impaired. Recently, an unexpected mechanism of renal magnesium wasting and hypomagnesemia was discovered by Adalat et al. (2). They first identified a teenager who had hepatocyte nuclear factor 1B (HNF 1B) mutation and presented with tetany and hypomagnesemia (0.620 mmol/L). Then they retrospectively reviewed 91 pediatric cases of renal malformation screened for HNF 1B mutation. Twenty-one had heterozygous mutations. Eight (44%) of 18 mutation carriers had hypomagnesemia (<0.65 mmol/L) compared with one (2%) of 48 of those without mutations. HNF 1B is a transcription factor that is expressed in developing kidneys, whereas, postnatally, proximal and distal tubules express the gene. Hnf 1b in mouse upregulates transcription of various genes whose human homologues are involved in inherited cystic kidney diseases. Adalat et al. (2) detected a highly conserved HNF 1B recognition site in FXYD2. They demonstrated HNF 1B–mediated transactivation of FXYD2. Thus, HNF 1B regulates transcription of FXYD2, which participates in the tubular handling of Mg2+. These results extend the phenotype of HNF 1B mutations to include hypomagnesemia. Serum Mg2+ levels should be measured in patients with HNF 1B mutations (2).

Molecular physiology of the thiazide-sensitive sodium–chloride cotransporter

This review summarizes recent advances in the understanding of the molecular physiology and regulation of the thiazide-sensitive sodium-chloride cotransporter (NCC). Mutations of with-no-lysine (WNK) kinases 1 and 4 result in hyperactivity of NCC and familial hyperkalemic hypertension, a genetic syndrome of hypertension. Recent studies have shown that WNK1 and WNK4 activate the STE20 family protein kinases Ste20-related proline/alanine-rich kinase and odd-skipped-related 1, resulting in phosphorylation and activation of NCC. Additionally, a mouse knock-in model for a WNK4 familial hyperkalemic hypertension mutant demonstrated increased Ste20-related proline/alanine-rich kinase/odd-skipped-related 1 and NCC phosphorylation. It is unclear how these studies fit with the data indicating that WNK4 inhibits NCC, and the familial hyperkalemic hypertension mutations of WNK4 are loss-of-function mutations. Another WNK kinase, WNK3, also regulates NCC, activating NCC and antagonizing the effect of WNK4. Extracellular signal-related kinase 1/2 mitogen-activated protein kinase activation by Ras guanyl nucleotide-releasing protein 1 is another kinase pathway that appears to be a potent regulator of NCC. Other studies have described a role for angiotensin II in pressure natriuresis via actions on NCC. Recent studies examining the hormonal regulation of NCC have implicated angiotensin II and aldosterone in regulation of the WNK4-Ste20-related proline/alanine-rich kinase-NCC pathway. NCC is subject to a complex regulatory network of kinases, which appear quite sensitive to alterations of the hormonal and physiologic milieu.

Gitelman Syndrome

Gitelman Syndrome

Gitelman's Syondrome: a Hereditary Disorder Characterized by Hypokalemia and Hypomagnesaemia

Gitelman’s syndrome is a type of hereditary tubular disorder, which is caused by inactive mutations in the gene, which encodes thiazide sensitive sodium chloride cotransporter (SLC12A3) in the distal convoluted tubule. Biochemical characteristic is comparable to effect of thiazide diuretics: hypokalemia, hypomagnesaemia, hypocalciuria, hypokalemic alkalosis and blood pressure is normal or lower. Gitelman’s syndrome is typically diagnosed accidentally (hypokalemia) in adolescents or adults and the course is benign. If present, the most prominent symptoms are muscular fatigue or occasional tetany. Treatment includes magnesium and potassium salts and potassium saving diuretics. We report here a case of an adult patient with Gitelman’s syndrome. The patient was a bearer of three heterozygote mutations in the gene, which encodes thiazide sensitive sodium chloride cotransporter (SLC12A3) in the distal convoluted tubule.

Upregulation of the thiazide‐sensitive sodium chloride cotransporter in the kidney is associated with the hypertension in D3 dopamine receptor heterozygous (D3 ‐/+) mice

D3 dopamine receptor (D3R) knockout (D3‐/‐) mice have hypertension related to an increased protein expression of thiazide‐sensitive sodium chloride cotransporter (NCC) in the renal distal convoluted tubule. D3R heterozygous mice (D3 ‐/+) are also hypertensive. To determine if a mechanism involved in the pathogenesis of hypertension in D3 ‐/+ is similar to that in D3‐/‐ mice, we measured renal NCC protein abundance in D3‐/+ and their wild type (D3+/+) littermates. On normal NaCl intake, blood pressure (BP, telemetry) was higher in D3‐/+ than in D3+/+ mice but body weight, serum electrolytes and urinary sodium excretion were similar. NCC protein expression in D3‐/+ mice was increased in the renal plasma membrane‐enriched (136±7) and intracellular‐vesicle enriched fractions (154±7) (% of D3+/+, n=6/group). To determine the role of the increased NCC expression in the hypertension of D3‐/+ mice, they were treated with hydrochlorothiazide (HTZ, 30 mg/Kg X 3 days, IP), a specific NCC inhibitor. There was a more than 2‐fold increase in HTZ‐induced natriuresis in D3‐/+ (19.2±3.0) relative to D3+/+ mice (8.1±2.3)(nEq/h, 6 hours post HTZ injection,n=5/group). BP decreased sooner (on day 2 in D3‐/+ mice and on day 3 in D3+/+ mice) and to a greater extent in D3‐/+ (21±2) than in D3+/+ mice (14±1)(mm Hg, n=4/group). We conclude that the hypertension of D3R deficient mice is associated with upregulation of the renal NCC.

Heterozygous mutations of the sodium chloride cotransporter in Chinese children: prevalence and association with blood pressure

Gitelman's syndrome (GS), which is caused by homozygous or compound heterozygous mutations of the thiazide-sensitive sodium chloride cotransporter (NCC), usually manifests in children and is associated with low blood pressure. However, the prevalence of heterozygous NCC mutations and their association with blood pressure in children have not yet been studied. Five hundred unrelated children from the Taipei Children Heart Study were enrolled. Genomic DNA was isolated from peripheral blood and the SLC12A3 gene was amplified by polymerase chain reaction (PCR). The 15 NCC mutations previously identified in Chinese patients with GS were evaluated using restriction fragment length polymorphism (RFLP) analysis. Blood pressure, biochemistry and urine pH were measured. The allelic frequency of heterozygous NCC mutations and their association with low blood pressure were also investigated. RFLP analysis for the 15 NCC mutations revealed heterozygous T60M in 1 child, T163M in 1, S283Y in 4, R642C in 2, W844X in 2, R928C in 9 and R959frameshift in 10 children. The overall incidence of positive heterozygous NCC mutations was approximately 2.9%. There were no significant differences in systolic or diastolic blood pressure, biochemical profiles or urine pH between children with heterozygous NCC mutations (n = 29) and non-affected controls (n = 471), except for slightly higher fasting plasma glucose concentrations in NCC-heterozygous children (91 +/- 2.3 versus 88 +/- 0.4 mg/dL, P < 0.05). Examination among the different NCC mutations showed that these children also had comparable blood pressures. We found a relatively high prevalence of heterozygous NCC mutations in Chinese children, suggesting that GS may not be rare in this population. Heterozygous NCC mutations were not associated with lower blood pressure in these Chinese children.

PTH suppresses sodium chloride cotransporter activity by a Mapk‐dependent process

Recently, we described that Diacylglycerol (DAG) analogues act through Ras guanyl‐releasing protein1 (RasGRP1) stimulation of ERK1/2 MAPK to decrease function and surface expression of the thiazide‐sensitive sodium chloride cotransporter (NCC). However, upstream hormonal mediators of DAG‐dependent NCC suppression have not been identified. Although PTH activates DAG through G‐protein coupled receptor activation of Phospholipase C, PTH regulation of NCC has not been demonstrated. Utilizing a mouse DCT (mDCT) cell line with native NCC activity, we investigated whether PTH may mediate suppression of NCC function through MAPK activation. Thiazide‐sensitive 22Na+ uptakes were measured by incubating mDCT cells for 15 minutes in a Cl−free medium before exposure to a 22Na+containing medium with or without thiazide. Uptakes were determined after 15 minute incubations with vehicle, PTH (10‐7M) alone, PTH + U0126 (MEK 1/2 MAPK inhibitor), and PTH + bisindolylmaleimide (BIM, PKC inhibitor). PTH decreased NCC activity by 43.6 +/− 4.5% (p&lt;0.05 for PTH vs. vehicle). U0126 blocked the PTH‐induced suppression while BIM had no effect on the suppression. This is consistent with our previous data that RasGRP1 and not PKC is the mediator of DAG effects on NCC activity. These studies indicate that PTH decreases NCC function via downstream activation of the MAPK pathway. This work was supported by NIH Grant K08 DK070668.