Sodium-chloride Cotransporter NCC Research Articles

Cryo-EM structure of the human sodium-chloride cotransporter NCC.

The sodium-chloride cotransporter NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure control. Here, we present the full-length structure of human NCC, with 2.9 Å for the transmembrane domain and 3.8 Å for the carboxyl-terminal domain. NCC adopts an inward-open conformation and a domain-swap dimeric assembly. Conserved ion binding sites among the cation-chloride cotransporters and the Na2 site are observed in our structure. A unique His residue in the substrate pocket in NCC potentially interacts with Na1 and Cl1 and might also mediate the coordination of Na2 through a Ser residue. Putative observed water molecules are indicated to participate in the coordination of ions and TM coupling. Together with transport activity assays, our structure provides the first glimpse of NCC and defines ion binding sites, promoting drug development for hypertension targeting on NCC.

The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter NCC but is not required for a potassium-induced reduction of NCC expression.

Na+ and K+ balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K+. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4–2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K+ on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In ex vivo experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K+ reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K+ induced reductions in NCC abundance.

Effects of Chronic Potassium Supplementation and the Accompanying Anion on Blood Pressure

Increased dietary NaCl is associated with higher blood pressure (BP), but dietary potassium supplementation can reduce BP and reduce ‘salt sensitivity’ of BP, improving cardiovascular (CV) outcomes. Rodents fed a high K+ diet often exhibit reduced activity of the sodium-chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. Recently, the effect of extreme K+ diets on rodent BP seemed inconsistent with patterns of BP modulation seen in humans. Therefore, the aim of this study was to establish how BP is altered during chronic alteration of dietary potassium intake alongside alternative anions and/or NaCl supplementation. Male mice implanted with telemetric BP monitors were initially normalized to a control diet of NaCl (0.3% Na+) and KCl (1.05% K+), and subsequently maintained on this control diet or a high NaCl (1.57% Na+) (HS) diet for 7 weeks. Subsequently, mice were stratified to receive either a high KCl (5.25% K+) or a high K-citrate (KCit, 5.25% K+) diet during control or high NaCl intake for a subsequent 3 weeks. Finally, a subset of mice were switched to a zero K+ diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cl- levels were monitored. 2 weeks 0 K+ diet significantly increased BP, an effect augmented under HS diet. A trend to reduce BP was apparent under control and HS diet during chronic KCl feeding (> effect during resting period), whereas chronic KCit feeding only tended to reduce BP during HS diet. Plasma K+ levels inversely correlated with BP, with chronic KCl inducing a significant increase of 0.5mM, whereas chronic KCit feeding had no significant effect. Plasma aldosterone was also significantly increased 3.5 fold by chronic KCl feeding but not significantly by KCit diet. Total and phosphorylated NCC levels were significantly increased on 0 K+ diet. KCl and KCit feeding during control NaCl or HS intake reduced total NCC levels, whereas phosphorylated NCC was only reduced during chronic KCl intake. All dietary conditions had minimal effect on levels of WNK/SPAK pathway proteins, but αENaC (cleaved/uncleaved) levels were increased, substantially more during KCit feeding. Pendrin abundance was also significantly elevated by KCit feeding. In summary, we show a negative correlation of BP with plasma K+, consistent with human interventional studies. Chronic KCl has a greater ability to lower BP than KCit feeding. This may be related to the ability of KCl to lower NCC phosphorylation, or that large increases in αENaC and pendrin levels during KCit feeding oppose the BP lowering effects of reduced total NCC. As aldosterone levels were increased more with KCl, a differential role of aldosterone also needs to be considered.

Magnesium and Calcium Homeostasis Depend on KCTD1 Function in the Distal Nephron

SUMMARYMagnesium (Mg2+) homeostasis depends on active transcellular Mg2+ reuptake from urine in distal convoluted tubules (DCTs) via the Mg2+ channel TRPM6, whose activity has been proposed to be regulated by EGF. Calcium (Ca2+) homeostasis depends on paracellular reabsorption in the thick ascending limbs of Henle (TALs). KCTD1 promotes terminal differentiation of TALs/DCTs, but how its deficiency affects urinary Mg2+ and Ca2+ reabsorption is unknown. Here, this study shows that DCT1-specific KCTD1 inactivation leads to hypomagnesemia despite normal TRPM6 levels because of reduced levels of the sodium chloride co-transporter NCC, whereas Mg2+ homeostasis does not depend on EGF. Moreover, KCTD1 deficiency impairs paracellular urinary Ca2+ and Mg2+ reabsorption in TALs because of reduced NKCC2/claudin-16/-19 and increased claudin-14 expression, leading to hypocalcemia and consequently to secondary hyperparathyroidism and progressive metabolic bone disease. Thus, KCTD1 regulates urinary reabsorption of Mg2+ and Ca2+ by inducing expression of NCC in DCTs and NKCC2/claudin-16/-19 in TALs.

The hypokalemia mystery: distinguishing Gitelman and Bartter syndromes from 'pseudo-Bartter syndrome'.

The Gitelman and Bartter syndromes (GS and BS, respectively) are characterized by the constellation of hypokalemia, hypochloremia, metabolic alkalosis, hyperreninemic hyperaldosteronism, low to normal blood pressure and juxtaglomerular apparatus hypertrophy. These are due to pathogenic variants in the genes that encode the thiazide-sensitive sodium–chloride cotransporter NCC (SLC12A3) in the distal convoluted tubule or transporters involved in sodium chloride reabsorption in the loop of Henle. ‘Pseudo-Bartter syndrome’ (PBS) is caused by extrarenal or acquired renal salt losses and shares the same plasma electrolyte profile and acid–base disturbances, making the distinction from the genetic forms very challenging. PBS has various etiologies, including diuretic and laxative abuse, self-induced vomiting and the side effects of some antimicrobials. Additionally, congenital chloride diarrhea, cystic fibrosis, Pendred syndrome and chloride-deficient diet can manifest as PBS. In this review we focus on factitious PBS secondary to diuretic abuse, laxative abuse or self-induced vomiting, whereby we point out some clinical and laboratory clues to help clinicians make the correct diagnosis.

Effects of Potassium Supplementation and the Accompanying Anion on Cardiovascular Parameters

In general, a high NaCl intake is associated with increased blood pressure (BP), whereas dietary potassium intake has a negative correlation with BP, cardiovascular (CV) outcomes and reduces the ‘salt sensitivity’ of BP. High K+ intake reduces activity of the sodium‐chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. This study sought to establish an independent role of the anion during chronic potassium supplementation on cardiovascular parameters. Male mice implanted with telemetric BP monitors were initially normalized to a control diet of NaCl (0.3% Na+) and KCl (1.05% K+), and subsequently maintained on this control diet or a high NaCl (1.57% Na+) diet for 7 weeks. Subsequently, mice were stratified to receive either a high KCl (5.25% K+) or a high K‐citrate (KCit, 5.25% K+) diet during control or high NaCl intake for a subsequent 3 weeks. Finally, a subset of mice were switched to a zero KCl diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cllevels were monitored.Fractional excretion (FE) of K+ was significantly increased in both high K+ diets after 3 weeks and was independent of NaCl load. KCl and KCit feeding significantly increased FE of Na+ similarly but did not augment an already increased FE Na+ in mice fed high NaCl. High NaCl intake alone had no significant effect on BP, and chronic high K+ diets did not significantly reduce BP. However, overall plasma K+ levels inversely correlated with BP. Interestingly, plasma Cl− levels also inversely correlated with BP. KCl fed mice exhibited lower heart rate relative to KCit fed animals. Regression analysis points to KCit fed mice having lower BP, of about 5–10mmHg, if at the same heart rate as KCl fed animals. In contrast, 2 weeks of low K+ diet increased BP, more notably in high NaCl fed animals (BP increase of 7mmHg). NaCl‐KCit fed mice exhibited a significant 14% reduction in heart weight tibia length ratio (HW:TL) compared to NaCl‐control fed animals. No significant reduction in HW:TL was evident from NaCl‐KCl fed mice.In summary, in the timeframe of this study, potassium supplementation had limited effects on BP in normotensive mice during normal or high NaCl intake despite increasing the FE Na+. However, chronic KCit feeding may have some benefits to cardiovascular health above those promoted by KCl. The molecular mechanisms underpinning these effects are under investigation.Support or Funding InformationThis work was funded by grants from Novo Nordisk Foundation, Danish Independent Research Council and the Leducq Foundation.

Inhibition of Protein Phosphatase 1 prevents high potassium mediated downregulation of the thiazide‐sensitive sodium chloride cotransporter NCC

The “aldosterone paradox” is a poorly understood physiological mechanism in which increases in the hormone aldosterone either serve to stabilize blood pressure by increasing sodium‐chloride (NaCl) reabsorption or to excrete potassium (K+). Altered NaCl transport via the NaCl cotransporter NCC in the renal distal convoluted tubule (DCT) has been suggested to play a central role in switching from one response to the other. Under low NaCl intake, NCC abundance and activity is high, whereas it is low during high K+ intake ‐ leading to increased delivery of NaCl to downstream segments and increased electrogenic K+ secretion. Despite its essential role, little is known about the molecular alterations in the DCT during the aldosterone paradox, and the proteins involved in the regulation of NCC during this response.To define the proteome of the DCT and how it is modified by increased circulating aldosterone levels subsequent to long term low dietary NaCl or high K+ intake, transgenic mice with expression of enhanced green fluorescent protein (eGFP) under the control of the parvalbumin promoter were used. These mice have specific eGFP‐labeling of DCT cells, allowing high purity fluorescence‐activated cell sorting (FACS) of DCT cells. Mice were maintained on low NaCl or high K+ diets for 4 days, which resulted in increased aldosterone levels and a change in NCC abundance. Following FACS, the DCT proteome was determined by liquid chromatography‐coupled tandem mass spectrometry. 3078 unique proteins were identified in the DCT. 210 proteins were significantly altered in abundance following a low NaCl diet. 625 were significantly altered in abundance following a high K+ diet. Protein Phosphatase 1α (Pp1α) abundance was only increased by high K+ diet. In freshly isolated mouse tubules from the renal cortex, a higher K+ concentration in the medium for 1–4 days resulted in a significant decrease in NCC expression and increased expression of PP1α, mimicking the in vivo situation. The K+‐induced decrease of total NCC levels could be inhibited by treating the cells with the PP1‐blocker tautomycetin, suggesting that PP1 is involved in the K+‐induced downregulation of NCC. In summary, these results suggest that PP1α is involved in the decreased NCC abundance seen during long term high potassium intake.Support or Funding InformationDanish Medical Research Council, Fondation LeDuq, Novo Nordisk Foundation, Aarhus University Research Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Urinary Extracellular Vesicle Protein Profiling and Endogenous Lithium Clearance Support Excessive Renal Sodium Wasting and Water Reabsorption in Thiazide-Induced Hyponatremia

IntroductionThiazide diuretics are among the most widely used antihypertensive medications worldwide. Thiazide-induced hyponatremia (TIH) is 1 of their most clinically significant adverse effects. A priori TIH must result from excessive saliuresis and/or water reabsorption. We hypothesized that pathways regulating the thiazide-sensitive sodium-chloride cotransporter NCC and the water channel aquaporin-2 (AQP2) may be involved. Our aim was to assess whether patients with TIH would show evidence of altered NCC and AQP2 expression in urinary extracellular vesicles (UEVs), and also whether abnormalities of renal sodium reabsorption would be evident using endogenous lithium clearance (ELC).MethodsBlood and urine samples were donated by patients admitted to hospital with acute symptomatic TIH, after recovery to normonatremia, and also from normonatremic controls on and off thiazides. Urinary extracellular vesicles were isolated and target proteins evaluated by western blotting and by nanoparticle tracking analysis. Endogenous lithium clearance was assessed by inductively coupled plasma mass spectrometry.ResultsAnalysis of UEVs by western blotting showed that patients with acute TIH displayed reduced total NCC and increased phospho-NCC and AQP2 relative to appropriate control groups; smaller differences in NCC and AQP2 expression persisted after recovery from TIH. These findings were confirmed by nanoparticle tracking analysis. Renal ELC was lower in acute TIH compared to that in controls and convalescent case patients.ConclusionReduced NCC expression and increased AQP2 expression would be expected to result in saliuresis and water reabsorption in TIH patients. This study raises the possibility that UEV analysis may be of diagnostic utility in less clear-cut cases of thiazide-associated hyponatremia, and may help to identify patients at risk for TIH before thiazide initiation.

Uromodulin is expressed in the distal convoluted tubule, where it is critical for regulation of the sodium chloride cotransporter NCC

Uromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the cotransporter NKCC2 and is involved in sodium chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent insitu hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal sodium chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones.

A mouse model of pseudohypoaldosteronism type II reveals a novel mechanism of renal tubular acidosis

Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in β-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.

EET enhances renal function in obese mice resulting in restoration of HO-1-Mfn1/2 signaling, and decrease in hypertension through inhibition of sodium chloride co-transporter

BackgroundWe have previously reported that epoxyeicosatrienoic acid (EET) has multiple beneficial effects on renal and adipose tissue function, in addition to its vasodilatory action; it increases insulin sensitivity and inhibits inflammation. In an examination of the signaling mechanisms by which EET reduces renal and peri-renal fat function, we hypothesized that EET ameliorates obesity-induced renal dysfunction by improving sodium excretion, reducing the sodium-chloride cotransporter NCC, lowering blood pressure, and enhancing mitochondrial and thermogenic gene levels in PGC-1α dependent mice. MethodsEET-agonist treatment normalized glucose metabolism, renal ENaC and NCC protein expression, urinary sodium excretion and blood pressure in obese (db/db) mice. A marked improvement in mitochondrial integrity, thermogenic genes, and PGC-1α−HO-1-adiponectin signaling occurred. Knockout of PGC-1α in EET-treated mice resulted in a reversal of these beneficial effects including a decrease in sodium excretion, elevation of blood pressure and an increase in the pro-inflammatory adipokine nephroblastoma overexpressed gene (NOV). In the elucidation of the effects of EET on peri-renal adipose tissue, EET increased adiponectin, mitochondrial integrity, thermogenic genes and decreased NOV, i.e. “Browning’ peri-renal adipose phenotype that occurs under high fat diets. Taken together, these data demonstrate a critical role of an EET agonist in the restoration of healthy adipose tissue with reduced release of inflammatory molecules, such as AngII and NOV, thereby preventing their detrimental impact on sodium absorption and NCC levels and the development of obesity-induced renal dysfunction.

The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na+ and K+ homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function.

SP022MOLECULAR CHARACTERIZATION OF A NOVEL MUTATION IN THE RENAL NACL COTRANSPORTER CAUSING GITELMAN'S SYNDROME BY IMPAIRING TRANSPORTER TRAFFICKING

Mutations affecting the sodium-chloride cotransporter (NCC) in the distal convoluted tubule of the nephron are causative of Gitelman'€™s syndrome (GS), a rare autosomal recessive disease characterized by electrolytic alterations similar to those induced by high dose thiazide treatment. Notably, the co-existence of hypomagnesemia and hypocalciuria is a feature of GS which is a distinguish hallmark from another hypokalemic renal tubulopathy, the Bartter'€™s syndrome (BS). Commonly GS is heterozygous compound with an estimated prevalence of 1:40000 and can be silent for years before the revealing in the early adulthood. Recognizing the genetic background is fundamental for the screening and the diagnosis of the disease, as recently studies showed that mutation affecting regulators of renal salt handling are underestimated in the general population. In a registry of BS/GS based at our University we discovered a novel point mutation (c.1204G>A which codify for an amino acid exchange G394D) in the sodium-chloride cotransporter NCC (SLC12A3) in a young woman with hypokalemia, hypomagnesemia and hypocalciuria associated to muscle pain and cramps. The present study aimed to investigate how this mutation affects NCC function by using a molecular biology approach and providing functional evidences. After a prior screening with bioinformatics tools predicting the possible pathogenicity of the mutation, were created different expression vectors with either the wild-type (wt-NCC) or the mutated G394D-NCC sequences. DNA and in-vitro transcribed RNA were afterwards transfected in a human embryonic kidney cells line (HEK293) and injected into oocytes deriving from Xenopus Laevis frog respectively. In transfected HEK 293 cells, wildtype NCC was detected by immunoblotting as two bands at approximately 130 kDa and 115 kDa corresponding to fully and core-glycosylated NCC, respectively. In contrast, G394D-NCC was seen as a single band at about 115 kDa only, suggesting an impaired maturation of the mutated protein. Similar findings were made in the oocyte expression system. Confocal microscopy on the oocytes, did also show a strong cell surface localization of wildtype NCC while mutated NCC was retained at intracellular compartments. Consistently, a decent thiazide-sensitive 22Na+ uptake into injected oocytes was only found for wildtype but not mutated NCC. Taken together all the findings in this study, a novel GS point mutation has been characterized to diminish NCC function by impairing trafficking of the protein to the cell surface. The absence of any mature glycosylation form of G394D-NCC suggests that the mutation impairs protein folding leading to a retention of NCC in the endoplasmic reticulum.

FGF23 regulation of renal tubular solute transport.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone known to suppress phosphate reabsorption in the kidney. The purpose of this review was to highlight the recent advances in the area of FGF23-regulated solute transport in the kidney. Recent evidence suggests that FGF23 suppresses phosphate reabsorption in renal proximal tubular epithelium by a Klotho-dependent, FGF receptor (FGFR)-1 and FGFR4-mediated signaling mechanism that may also involve Janus kinase 3. Moreover, it was recently established that FGF23 signaling in the distal renal tubule targets with-no-lysine kinase-4 (WNK4), a key molecule in the regulation of solute transport in the distal nephron. By targeting WNK4, FGF23 has been shown to increase the membrane abundance of the epithelial calcium channel TRPV5 and of the sodium-chloride cotransporter NCC, resulting in augmented renal calcium and sodium reabsorption. Significant progress has been made in the further characterization of the signaling pathways involved in the FGF23-induced inhibition of phosphate transport in proximal tubular epithelium, and major new functions of FGF23 in solute transport have been discovered in distal renal tubules. The calcium- and sodium-conserving functions of FGF23 may have major implications for the pathophysiology of cardiovascular diseases. VIDEO ABSTRACT.

Vasopressin regulation of sodium transport in the distal nephron and collecting duct.

Arginine vasopressin (AVP) is released from the posterior pituitary gland during states of hyperosmolality or hypovolemia. AVP is a peptide hormone, with antidiuretic and antinatriuretic properties. It allows the kidneys to increase body water retention predominantly by increasing the cell surface expression of aquaporin water channels in the collecting duct alongside increasing the osmotic driving forces for water reabsorption. The antinatriuretic effects of AVP are mediated by the regulation of sodium transport throughout the distal nephron, from the thick ascending limb through to the collecting duct, which in turn partially facilitates osmotic movement of water. In this review, we will discuss the regulatory role of AVP in sodium transport and summarize the effects of AVP on various molecular targets, including the sodium-potassium-chloride cotransporter NKCC2, the thiazide-sensitive sodium-chloride cotransporter NCC, and the epithelial sodium channel ENaC.

Integrated compensatory network is activated in the absence of NCC phosphorylation.

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

P2Y2 receptor activation inhibits the expression of the sodium-chloride cotransporter NCC in distal convoluted tubule cells

Luminal nucleotide stimulation is known to reduce Na(+) transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca(2+) transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca(2+) transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca(2+) concentration ([Ca(2+)]i) may control NCC transcription, we overexpressed the Ca(2+)-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca(2+). Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from -1 to -2,200bp was not regulated by changes in [Ca(2+)]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca(2+) signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca(2+) concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.

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.