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Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in genes that regulate activity of the renal sodium chloride cotransporter (NCC), including the kinases WNK1 and WNK4, and Cullin3 (Cul3) and KLHL3, members of a ring ubiquitin ligase complex. To examine the physiological roles of Cul3 and how mutations in it cause FHHt, we generated Cul3-/- mice. An inducible system that disrupts Cul3 specifically along the entire nephron was used since complete Cul3 disruption is embryonic lethal. Western blots showed nearly complete absence of Cul3 in Cul3-/- kidneys. Cul3-/- mice displayed volume contraction, hypochloremia and alkalosis, and elevated plasma aldosterone. Blood pressure, measured by radiotelemetry, was similar in both groups on normal NaCl diet, but during NaCl restriction, Cul3-/- mice displayed a progressive reduction in blood pressure. After 9 days, the 24h mean blood pressure pressure was 18 mmHg lower in Cul3-/- mice. Expression of total and phospho-NCC were slightly elevated in Cul3-...

First published August 8, 2001; 10.1152/ajprenal.00148.2001.—The mammalian distal nephron develops a complex assembly of specialized cell types to accomplish the fine adjustment of urinary electrolyte composition. The epithelia of the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) show an axial structural heterogeneity that has been functionally elucidated by the localization of proteins involved in transepithelial ion transport. We compared the distribution of the thiazide-sensitive Na+-Cl−cotransporter (TSC), basolateral Na+/Ca2+exchanger (Na/Ca), cytosolic calcium-binding proteins calbindin D28Kand parvalbumin, and the key enzyme for selective aldosterone actions, 11β-hydroxysteroid-dehydrogenase 2 (11HSD2), in the distal convolutions of the mouse. In the mouse, as opposed to the rat, we found no clear subsegmentation of the DCT into a proximal (DCT1) and a distal (DCT2) portion. The TSC was expressed along the entire DCT. Na/Ca and calbindin D28Kwere similarly expressed along most of the DCT, with minor exceptions in the initial portion of the DCT. Both were also present in the CNT. Parvalbumin was found in the entire DCT, with an occasional absence from short end portions of the DCT, and was not present in CNT. 11HSD2 was predominantly located in the CNT and CCD. Short end portions of DCT only occasionally showed the 11HSD2 signal. We also observed an overlap of 11HSD2 immunoreactivity and mRNA staining. Our observations will have implications in understanding the physiological effects of gene disruption and targeting experiments in the mouse.

Chronic infusion of loop diuretics into animals induces structural and functional changes in the distal nephron. These changes include increases in the activity of the thiazide-sensitive Na(+)/Cl(-)-cotransporter (NCC). The NCC was recently demonstrated to be an aldosterone-induced protein. These experiments were designed to test the hypotheses that chronic loop diuretic infusion, with replacement of NaCl losses, increases NCC protein abundance and that this effect results, in part, from stimulation by aldosterone. Sprague-Dawley rats received vehicle (group 1), furosemide (22 mg/100 g body wt per d) (group 2), or furosemide plus spironolactone (22 and 20 mg/100 g body wt per d, respectively) (group 3). Urine output was higher for groups 2 and 3 than for group 1 (151 +/- 32, 149 +/- 24, and 12 +/- 4 ml, respectively; P0.0001). Immunoblot analysis of NCC protein demonstrated that loop diuretics increased NCC protein abundance by nearly 100% (from 2562 +/- 30 to 5248 +/- 151 arbitrary units, P0.01). Spironolactone decreased NCC protein abundance by 66% (to 3532 +/- 113 units), compared with the furosemide-treated group (P0.005). Northern blot analysis of NCC mRNA demonstrated no significant effect of furosemide (NCC/glyceraldehyde-3-phosphate dehydrogenase ratios: group 1, 0.6 +/- 0.12; group 2, 0.5 +/- 0.05; P0.05, NS) These results indicate that increased NCC activity during chronic loop diuretic infusion is associated with increases in NCC protein abundance. A portion of the furosemide effect can be prevented by blockade of mineralocorticoid receptors.

The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and arterial BP. Loss-of-function mutations of NCC cause renal salt wasting with arterial hypotension (Gitelman syndrome). Conversely, mutations in the NCC-regulating WNK kinases or kelch-like 3 protein cause familial hyperkalemic hypertension. Here, we performed automated sorting of mouse DCTs and microarray analysis for comprehensive identification of novel DCT-enriched gene products, which may potentially regulate DCT and NCC function. This approach identified protein phosphatase 1 inhibitor-1 (I-1) as a DCT-enriched transcript, and immunohistochemistry revealed I-1 expression in mouse and human DCTs and thick ascending limbs. In heterologous expression systems, coexpression of NCC with I-1 increased thiazide-dependent Na(+) uptake, whereas RNAi-mediated knockdown of endogenous I-1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I-1 (I-1(-/-)) knockout mice without changes in total NCC expression. The abundance and phosphorylation of other renal sodium-transporting proteins, including NaPi-IIa, NKCC2, and ENaC, did not change, although the abundance of pendrin increased in these mice. The abundance, phosphorylation, and subcellular localization of SPAK were similar in wild-type (WT) and I-1(-/-) mice. Compared with WT mice, I-1(-/-) mice exhibited significantly lower arterial BP but did not display other metabolic features of NCC dysregulation. Thus, I-1 is a DCT-enriched gene product that controls arterial BP, possibly through regulation of NCC activity.

A paper recently published by Groenestege and colleagues [8] used positional cloning to determine the cause of a rare inherited magnesium-wasting syndrome, autosomal recessive renal hypomagnesemia. The results showed that certain mutations in the epidermal growth factor (EGF) gene cause this disease. This suggests, perhaps surprisingly, that EGF and its receptor comprise a previously unrecognized signaling pathway in the human kidney that participates importantly in magnesium homeostasis. The EGF gene is highly expressed along the distal convoluted tubule (DCT), an important site for regulating urinary magnesium excretion. The product of the gene is a large membrane-bound molecule, expressed at both the apical and basolateral surfaces. A portion of the extracellular domain can be cleaved to form the 53-amino-acid hormone, EGF; it is believed that cleavage occurs preferentially at the basolateral surface, leading to increased apparent EGF abundance at the apical membrane. In the DCT, EGF binds to receptors at the basolateral surface, making basolateral EGF expression a necessary prerequisite for signaling. The gene defect that causes autosomal recessive renal hypomagnesemia appears to disrupt trafficking of pro-EGF to the basolateral membrane, thereby impeding its signaling capability. The results also showed that EGF stimulates magnesium transport by TRPM6 (transient receptor potential cation channel, subfamily M, member 6), a channel that is expressed at the apical membrane of DCT cells and appears to be a primary path for apical magnesium entry. Interestingly, the investigators also corroborated previous reports that cancer patients treated with an antagonist of the EGF receptor, cetuximab, develop renal magnesium wasting and hypomagnesemia, emphasizing the significance of EGF in maintaining magnesium balance in humans.

Rabbit distal convoluted tubule coexpresses NaCl cotransporter and 11β-hydroxysteroid dehydrogenase 2 mRNA. Background Although the renal cortical collecting duct (CCD) is a principal target for aldosterone, recent evidence suggests that salt transport by other nephron segments may also be regulated by aldosterone. Electroneutral and thiazide-sensitive NaCl cotransport by the distal convoluted tubule (DCT) of the rat is increased in animals deprived of dietary NaCl. We tested the hypothesis that the DCT of the rabbit is an aldosterone target tissue. Methods The single-nephron reverse-transcriptase/polymerase chain reaction (RT-PCR) technique was used to determine mRNA expression of NaCl cotransporter and 11β-HSD 2 in dissected nephron segments. The rabbit NaCl cotransporter was first cloned and rabbit-specific primers selected. A micro-assay was developed to assess 11β-HSD 2 enzyme activity in 0.5mm samples of the same nephron segments. Results NaCl cotransporter was expressed in 0 of 6 proximal tubule (PT), 6 of 6 DCT and 3 of 6 CCD samples, while 11β-HSD was found in 0 of 7 PT, 7 of 7 DCT and 9 of 9 CCD samples. Corticosterone was converted to 11-dehydrocorticosterone at a high rate and to a similar extent by both the DCT and CCD, but not the PT. Conclusions We conclude that the DCT is a target tissue for the action of aldosterone. Axial heterogeneity of electroneutral (in DCT) and electrogenic (in CCD) Na transporters along the distal nephron may improve sodium recovery in low salt and volume states.

Mineralocorticoid hormones regulate salt transport along the distal nephron by binding to intracellular receptors and activating gene transcription. Previous experiments showed that systemic aldosterone infusions stimulate thiazide-sensitive Na and Cl transport by distal convoluted tubule (DCT) cells; this effect could have been direct or secondary to systemic hormonal effects. Aldosterone target tissues express both mineralocorticoid receptors and the metabolic enzyme 11beta-hydroxysteroid dehydrogenase type 2. Mineralocorticoid receptors have been localized to the DCT in some experiments, but not in others. Expression of 11beta-hydroxysteroid dehydrogenase type 2 by DCT cells has not been investigated. The present experiments were designed to test the hypothesis that rat DCT cells are targets of aldosterone action. Patterns of mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase, thiazide-sensitive Na-Cl cotransporter, and Na/Ca exchanger expression along the distal tubule were examined. A polyclonal antibody was generated to localize the thiazide-sensitive Na-Cl cotransporter. Thiazide-sensitive Na-Cl cotransporter and 11beta-hydroxysteroid dehydrogenase expression were examined using both in situ hybridization and immunocytochemistry; Na/Ca exchanger and mineralocorticoid receptor expression were examined by immunocytochemistry. The results indicate that 11beta-hydroxysteroid dehydrogenase is expressed by DCT cells, as well as connecting tubule cells and principal cells of the collecting duct; expression levels are low near the junction with the thick ascending limb and rise near the transition to the connecting tubule. Mineralocorticoid receptors are expressed by DCT cells, as well as along the thick ascending limb, connecting tubule, and collecting duct. The results indicate that components of the mineralocorticoid receptor system are expressed by DCT cells, suggesting that these cells are targets of aldosterone action.

Liddle syndrome is monogenic hypertension caused by mutations in the epithelial Na+ channel (ENaC) that interfere with its ubiquitylation by Nedd4-2. In this issue, Ronzaud and colleagues found that deleting Nedd4-2 from kidney tubules in adult mice led to ENaC accumulation, but not at the plasma membrane, as predicted from current models. Instead, abundance of the sodium chloride transporter NCC increased at the plasma membrane, and the mice have some features of increased NCC activity. Together, the results suggest that defective ubiquitylation of ENaC by Nedd4-2 may not fully explain Liddle syndrome and that Nedd4-2 modulates NCC more strongly.

The term diuretic derives from the Greek diouretikos , meaning “to promote urine.” Although infusion of saline or ingestion of water would therefore qualify as being diuretic, the term diuretic usually connotes a drug that can reduce the extracellular fluid (ECF) volume by increasing urinary solute or water excretion. The term aquaretic has sometimes been applied to drugs that increase excretion of solute free water, distinguishing them from traditional diuretics, which increase solute and water together. The clinical picture of ECF volume expansion leading to edema or “dropsy” (from the Latin, hydrops ) has been recognized since the earliest days of recorded history. Ancient Egyptians referred to “flooding of the heart,” and the Hippocratic Corpus later suggested specific remedies for dropsical patients, although their results are not noted. In 1553, Paracelsus recorded the first truly effective form of therapy for dropsy, inorganic mercury (Calomel). Inorganic mercury remained the mainstay of diuretic treatment until the beginning of this century.

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na+–Cl− co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr53 NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.

The purpose of this review is to summarize the available information regarding salt sensitivity particularly as it relates to non-Hispanic blacks and Hispanics and to clarify possible etiologies, especially those that might shed light on potential treatment options. In non-Hispanic blacks, there is evidence that endothelial dysfunction, reduced potassium intake, decreased urinary kallikrein excretion, upregulation of sodium channel activity, dysfunction in atrial natriuretic peptide (ANP) production, and APOL1 gene nephropathy risk variants may cause or contribute to salt sensitivity. Supported treatment avenues include diets high in potassium and soybean protein, the components of which stimulate nitric oxide production. Racial heterogeneity complicates the study of salt sensitivity in Hispanic populations. Caribbean Hispanics, who have a higher proportion of African ancestry, may respond to commonly prescribed anti-hypertensive agents in a way that is characteristic of non-Hispanic black hypertensives. The low-renin hypertensive phenotype commonly seen in non-Hispanic blacks has been linked to salt sensitivity and may indicate an increased risk for salt sensitivity in a portion of the Hispanic population. In conclusion, increased morbidity and mortality associated with salt sensitivity mandates further studies evaluating the efficacy of tailored dietary and pharmacologic treatment in non-Hispanic blacks and determining the prevalence of low renin hypertension and salt sensitivity within the various subgroups of Hispanic Americans.

An electroneutral thiazide-sensitive Na-Cl cotransport pathway (TSC) has been localized functionally to the distal convoluted tubule (DCT), although the TSC has also been detected in the connecting tubule (CNT), the cortical collecting duct, and the medullary collecting tubule as well. The present experiments were designed to localize expression of message for the TSC in rat and human kidney. A riboprobe, generated from the mouse TSC, was used for in situ hybridization. Simultaneous immunocytochemistry, using antibodies to Tamm-Horsfall protein, band 3, and the Na+/Ca2+ exchanger, permitted delineation of specific nephron segments. In rat, message for the TSC was highly expressed in DCT cells but not elsewhere. The transition from thick ascending limb to DCT was abrupt, whereas the transition to CNT was gradual. In the more distal region of rat DCT (DCT-2), which contained few intercalated cells, both TSC message and Na+/Ca2+ exchanger immunoreactivity were present. Treatment of rats with furosemide for 5 days increased expression of TSC message within the DCT but did not induce its expression elsewhere. In humans, expression of TSC message was also highest in cells of the DCT. In humans, however, expression extended well into the CNT. These experiments indicate that the TSC is expressed predominantly by DCT cells in both rat and humans, although expression extends into the CNT cells in humans. They also show that the TSC and Na+/Ca2+ exchanger are coexpressed by a subpopulation of DCT cells near the junction with the CNT.

The greatest breakthrough in the history of the drug treatment of hypertension came with the discovery of the orally effective diuretic, chlorothiazide ([1][1]). Hypertension affects up to 25% of the adult population in industrialized countries. It contributes importantly to morbidity and

Neuroblastoma is diagnosed over a wide age range from birth through young adulthood, and older age at diagnosis is associated with a decline in survivability.To identify genetic mutations that are associated with age at diagnosis in patients with metastatic neuroblastoma.Whole genome sequencing was performed on DNA from diagnostic tumors and their matched germlines from 40 patients with metastatic neuroblastoma obtained between 1987 and 2009. Age groups at diagnosis included infants (0-18 months), children (18 months-12 years), and adolescents and young adults (≥12 years). To confirm the findings from this discovery cohort, validation testing using tumors from an additional 64 patients obtained between 1985 and 2009 also was performed. Formalin-fixed, paraffin-embedded tumor tissue was used for immunohistochemistry and fluorescence in situ hybridization. Telomere lengths were analyzed using whole genome sequencing data, quantitative polymerase chain reaction, and fluorescent in situ hybridization.Somatic recurrent mutations in tumors from patients with neuroblastoma correlated with the age at diagnosis and telomere length.In the discovery cohort (n = 40), mutations in the ATRX gene were identified in 100% (95% CI, 50%-100%) of tumors from patients in the adolescent and young adult group (5 of 5), in 17% (95% CI, 7%-36%) of tumors from children (5 of 29), and 0% (95% CI, 0%-40%) of tumors from infants (0 of 6). In the validation cohort (n = 64), mutations in the ATRX gene were identified in 33% (95% CI, 17%-54%) of tumors from patients in the adolescent and young adult group (9 of 27), in 16% (95% CI, 6%-35%) of tumors from children (4 of 25), and in 0% (95% CI, 0%-24%) of tumors from infants (0 of 12). In both cohorts (N = 104), mutations in the ATRX gene were identified in 44% (95% CI, 28%-62%) of tumors from patients in the adolescent and young adult group (14 of 32), in 17% (95% CI, 9%-29%) of tumors from children (9 of 54), and in 0% (95% CI, 0%-17%) of tumors from infants (0 of 18). ATRX mutations were associated with an absence of the ATRX protein in the nucleus and with long telomeres.ATRX mutations were associated with age at diagnosis in children and young adults with stage 4 neuroblastoma.clinicaltrials.gov Identifier: NCT00588068.

Despite wide variations in dietary NaCl intake, homeostatic mechanisms ensure that renal NaCl excretion matches intake at steady state. This does not imply, however, that extracellular fluid volume is maintained within narrow limits. In contrast with blood pressure, which appears to be tightly controlled, extracellular fluid volume varies significantly, even in normal individuals, when dietary NaCl intake changes. Cardiac, liver, or renal disease can perturb the relationship between NaCl intake and extracellular fluid volume and lead to symptomatic edema. All major classes of diuretic drugs in use today were developed between 1950 and 1970. These drugs were developed empirically, without knowledge of specific ion transport pathways, but experimental work during the past 15 years has shown that each major class of diuretic inhibits a specific ion transport protein in the kidney. These transport proteins have been characterized physiologically and the mechanisms by which each diuretic drug inhibits ion transport have been defined. Antibodies directed against these transport proteins have localized ion transport pathways to specific cell types along the nephron. Most recently, isoforms of each class of diuretic-sensitive Na transport pathway have been cloned. Ongoing experimental work is aimed at exploring relationships between families of transporters, determining the structural prerequisites for ion transport, and studying molecular mechanisms of transport regulation. Treatment of edema with diuretics is often straightforward, but can lead to adaptive changes in nephron structure and function. These adaptations can limit the effectiveness of diuretic drugs; maneuvers aimed at blocking these processes can be effective approaches to patients who are resistant to diuretic drugs.

The clinical use of diuretics almost uniformly predated the localization of their site of action. The consequence of diuretic specificity predicts clinical application and side effect, and the proximity of the sodium transporters, one to the next, often dictates potency or diuretic efficiency. All diuretics function by inhibiting the normal transport of sodium from the filtrate into the renal tubular cells. This movement of sodium into the renal epithelial cells on the apical side is facilitated by a series of transporters whose function is, in turn, dependent on the adenosine triphosphate (ATP)-dependent Na-K cotransporter on the basolateral side of the cell. Our growing understanding of the physiology of sodium transport has spawned new possibilities for diuretic development.

BACKGROUND For vendor-derived mice, an acclimation period of 1 week is usually recommended before blood pressure measurements are started. However, we observed hypertension in wild-type vendor-derived mice 1 week after shipping. METHODS The index group (n = 12, BALB/c, age 3 months, weight 26-28 g) was shipped overnight (by truck, duration 13 h).Tail-cuff systolic blood pressures (SBPs) of the index group were compared to two control groups (n = 6/group), one acclimated for 3 weeks after shipping, and one derived from an in-house colony. RESULTS One week after shipping, SBP in the index group was 141 +/- 3 mm Hg. Because this was much higher than reported previously for this strain, acclimation was prolonged. Six weeks after shipping, SBP had fallen to 124 3 mm Hg (P < 0.005). During this time, heart rate also fell from 721 +/- 15 to 665 +/- 13 bpm (P < 0.01). SBP in the two control groups was also lower than in the index group 1 week after shipping, including the group acclimated for 3 weeks (129 +/- 3 vs. 141 +/- 3 mm Hg, P < 0.05) and the in-house mice (124 +/- 3 vs. 141 +/- 3 mm Hg, P < 0.005). CONCLUSIONS Vendor-derived mice are hypertensive 1 week after shipping, become normotensive after 3 weeks, but do not return to levels of in-house mice until after 6 weeks. Acclimation periods of at least 3 weeks are required when measuring blood pressure in mice.

WNKs are serine/threonine kinases that comprise a unique branch of the kinome. They are so-named owing to the unusual placement of an essential catalytic lysine. WNKs have now been identified in diverse organisms. In humans and other mammals, four genes encode WNKs. WNKs are widely expressed at the message level, although data on protein expression is more limited. Soon after the WNKs were identified, mutations in genes encoding WNK1 and -4 were determined to cause the human disease familial hyperkalemic hypertension (also known as pseudohypoaldosteronism II, or Gordon's Syndrome). For this reason, a major focus of investigation has been to dissect the role of WNK kinases in renal regulation of ion transport. More recently, a different mutation in WNK1 was identified as the cause of hereditary sensory and autonomic neuropathy type II, an early-onset autosomal disease of peripheral sensory nerves. Thus the WNKs represent an important family of potential targets for the treatment of human disease, and further elucidation of their physiological actions outside of the kidney and brain is necessary. In this review, we describe the gene structure and mechanisms regulating expression and activity of the WNKs. Subsequently, we outline substrates and targets of WNKs as well as effects of WNKs on cellular physiology, both in the kidney and elsewhere. Next, consequences of these effects on integrated physiological function are outlined. Finally, we discuss the known and putative pathophysiological relevance of the WNKs.

Udgivelsesdato: 2010-Apr-12 Proper control of NaCl excretion in the kidney is central to bodily functions. Yet, many mechanisms that regulate reabsorption of sodium and chloride in the kidney remain incompletely understood. Here, we identified an important role played by the intracellular sorting receptor SORLA (sorting protein-related receptor with A-type repeats) in functional activation of renal ion transporters. We demonstrate that SORLA is expressed in epithelial cells of the thick ascending limb (TAL) of Henle's loop and that lack of receptor expression in this cell type in SORLA-deficient mice results in the inability to properly reabsorb sodium and chloride during osmotic stress. The underlying cellular defect was correlated with an inability of the TAL to phosphorylate Na(+)-K(+)-Cl(-) cotransporter (NKCC) 2, the major sodium transporter in the distal nephron. SORLA functionally interacts with Ste-20-related proline-alanine-rich kinase (SPAK), an activator of NKCC2, and receptor deficiency is associated with mis-sorting of SPAK. Our data suggests a novel regulatory pathway whereby intracellular trafficking of SPAK by the sorting receptor SORLA is crucial for proper NKCC2 activation, and for maintenance of renal ion balance.

Aldosterone regulates volume homeostasis and blood pressure by enhancing sodium reabsorption in the kidney’s distal nephron (DN). On the apical surface of these renal epithelia, aldosterone increases expression and activity of the thiazide-sensitive Na-Cl cotransporter (NCC) and the epithelial sodium channel (ENaC). While the cellular mechanisms by which aldosterone regulates ENaC have been well characterized, the molecular mechanisms that link aldosterone to NCC-mediated Na+/Cl– reabsorption remain elusive. The serine/threonine kinase with-no-lysine 4 (WNK4) has previously been shown to reduce cell surface expression of NCC. Here we measured sodium uptake in a Xenopus oocyte expression system and found that serum and glucocorticoid–induced kinase 1 (SGK1), an aldosterone-responsive gene expressed in the DN, attenuated the inhibitory effect of WNK4 on NCC activity. In addition, we showed — both in vitro and in a human kidney cell line — that SGK1 bound and phosphorylated WNK4. We found one serine located within an established SGK1 consensus target sequence, and the other within a motif that was, to our knowledge, previously uncharacterized. Mutation of these target serines to aspartate, in order to mimic phosphorylation, attenuated the effect of WNK4 on NCC activity in the Xenopus oocyte system. These data thus delineate what we believe to be a novel mechanism for aldosterone activation of NCC through SGK1 signaling of WNK4 kinase.

With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg2+ levels, with a prominent function for the Mg2+-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg2+ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K+) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K+ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg2+ reabsorption.

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.

Diuretics (see Table 1.1 for a physiological classification) remain an important part of the medical therapy for patients with congestive heart failure (CHF). They control fluid retention and rapidly relieve the congestive symptoms of heart failure (HF). The American College of Cardiology/American Heart Association assigned them a class I indication in patients with symptomatic heart failure who have evidence of fluid retention [1]. Indeed, diuretics are the only drugs used in the treatment of HF that control fluid retention and that rapidly produce symptomatic benefits in patients with pulmonary and/or peripheral edema. Because diuretics alone are unable to effect clinical stability in patients with HF, they should always be used in combination with an angiotensin converting enzyme (ACE) inhibitor and a β-blocker. Despite the widespread use of diuretics, there have yet to be large randomized clinical trials that evaluate their effects on mortality or morbidity (with the exception of aldosterone antagonists, which will be considered separately). Furthermore, care must be exercised in the use of diuretics as both hypovolemia secondary to over-diuresis and hypervolemia secondary to under-diuresis have profound effects on cardiac pathophysiology. Therefore, questions remain about appropriate diuretic use [2]. This chapter will explore the effects, pharmacokinetics, and clinical utility of diuretics in patients with congestive heart failure. Vascular effects of diuretics

The pathogenesis of essential hypertension remains unknown, but thiazide diuretics are frequently recommended as first-line treatment. Recently, familial hyperkalemic hypertension (FHHt) was shown to result from activation of the thiazide-sensitive Na-Cl cotransporter (NCC) by mutations in WNK4, although the mechanism for this effect remains unknown. WNK kinases are unique members of the human kinome, intimately involved in maintaining electrolyte balance across cell membranes and epithelia. Previous work showed that WNK1, WNK4, and a kidney-specific isoform of WNK1 interact to regulate NCC activity, suggesting that WNK kinases form a signaling complex. Here, we report that WNK3, another member of the WNK kinase family expressed by distal tubule cells, interacts with WNK4 and WNK1 to regulate NCC in both human kidney cells and Xenopus oocytes, further supporting the WNK signaling complex hypothesis. We demonstrate that physiological regulation of NCC in oocytes results from antagonism between WNK3 and WNK4 and that FHHt-causing WNK4 mutations exert a dominant-negative effect on wild-type (WT) WNK4 to mimic a state of WNK3 excess. The results provide a mechanistic explanation for the divergent effects of WT and FHHt-mutant WNK4 on NCC activity, and for the dominant nature of FHHt in humans and genetically modified mice.

With-No-Lysine [K] (WNKs) are a recently discovered family of serine/threonine protein kinases that contain a uniquely structured catalytic domain. Mutations in the genes encoding two family members, WNK1 and WNK4, cause a chloride-dependent, thiazide-sensitive inherited syndrome of hypertension and hyperkalemia. Over the past 5 years, physiologic studies have demonstrated that these proteins regulate transcellular and paracellular epithelial ion flux. In this mini review, we discuss WNK1 and WNK4 gene products and their regulatory effects on sodium chloride and potassium handling in the aldosterone-sensitive distal nephron. Experimental observations regarding the effects of these proteins on transport processes mediated by the thiazide-sensitive Na–Cl co-transporter, the epithelial sodium channel, the renal outer medullary potassium channel, and the paracellular pathway integrate into a model that suggests an essential role for WNKs in coordinating renal Na–Cl reabsorption and K + secretion.

The mechanisms that govern homeostasis of complex systems have been elusive but can be illuminated by mutations that disrupt system behavior. Mutations in the gene encoding the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and hyperkalemia. We show that physiology in mice transgenic for genomic segments harboring wild-type (TgWnk4(WT)) or PHAII mutant (TgWnk4(PHAII)) Wnk4 is changed in opposite directions: TgWnk4(PHAII) mice have higher blood pressure, hyperkalemia, hypercalciuria and marked hyperplasia of the distal convoluted tubule (DCT), whereas the opposite is true in TgWnk4(WT) mice. Genetic deficiency for the Na-Cl cotransporter of the DCT (NCC) reverses phenotypes seen in TgWnk4(PHAII) mice, demonstrating that the effects of the PHAII mutation are due to altered NCC activity. These findings establish that Wnk4 is a molecular switch that regulates the balance between NaCl reabsorption and K+ secretion by altering the mass and function of the DCT through its effect on NCC.

With-no-lysine kinase-1 (WNK1) gene mutations cause familial hyperkalemic hypertension (FHHt), a Mendelian disorder of excessive renal Na+ and K+ retention. Through its catalytic activity, full-length kinase-sufficient WNK1 (L-WNK1) suppresses its paralog, WNK4, thereby upregulating thiazide-sensitive Na-Cl cotransporter (NCC) activity. The predominant renal WNK1 isoform, KS-WNK1, expressed exclusively and at high levels in distal nephron, is a shorter kinase-defective product; the function of KS-WNK1 must therefore be kinase independent. Here, we report a novel role for KS-WNK1 as a dominant-negative regulator of L-WNK1. Na+ transport studies in Xenopus laevis oocytes demonstrate that KS-WNK1 downregulates NCC activity indirectly, by inhibiting L-WNK1. KS-WNK1 also associates with L-WNK1 in protein complexes in oocytes and attenuates L-WNK1 kinase activity in vitro. These observations suggest that KS-WNK1 plays an essential role in the renal molecular switch regulating Na+ and K+ balance; they provide insight into the kidney-specific phenotype of FHHt.

With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt.