Cotransporter NCC Research Articles

The interplay between calcineurin inhibitors (CNIs) and WNK bodies

KS-WNK1 is a kidney-specific scaffolding isoform of the With-No-Lysine (WNK) kinase signaling pathway. It is the predominant WNK1 isoform in the kidney, lacks a functional kinase domain, and is highly expressed in the distal convoluted tubule (DCT) where it drives the formation of biomolecular condensates called WNK bodies. During hypokalemia, WNK bodies bring together KS-WNK1, L-WNK1, WNK4, and SPAK, facilitating activation of the kinase cascade. This leads to increased downstream phosphorylation of the SLC12 cotransporter NCC. Mechanisms governing the reversibility and size of WNK body condensate scaffolds are poorly understood. We propose that phosphatases regulate KS-WNK1 and WNK body formation--a likely candidate being the phosphatase calcineurin (PP3). Prior work suggests that calcineurin dephosphorylates and inhibits the WNK-SPAK-NCC pathway, whereas calcineurin inhibitors (CNIs) such as tacrolimus increase phosphorylation and activation of the pathway. Here, we tested the hypothesis that CNIs also increase the abundance of KS-WNK1, causing increased WNK body size and abundance, and activation of the WNK-SPAK-NCC pathway. To study the effect of endogenous WNK kinases on KS-WNK1 function, WT HEK293 cells or HEK293 L-WNK1 KO cells were transiently transfected with KS-WNK1-HA-mRuby, treated with tacrolimus and studied by immunofluorescence confocal microscopy and immunoblotting of supernatant and detergent-insoluble pellet fractions. In both WT and KO cells, tacrolimus significantly increased WNK body abundance and size. Consistent with its localization within WNK bodies, KS-WNK1 protein was enriched in the pellet fraction by 10-fold in both WT and KO cells. KS-WNK1 protein abundance was increased by tacrolimus in a dose- (0-400ng/ml) and time-dependent manner (0-72h), an effect that was independent of endogenous L-WNK1 expression. Consistent with previous studies, SPAK abundance and phosphorylation was increased with tacrolimus treatment in a dose and time-dependent manner. Interestingly, SPAK localization was shifted into the pellet fraction in WT cells expressing KS-WNK1, whereas it was localized in the supernatant in L-WNK1 KO cells. The tacrolimus-induced increase in KS-WNK1 and pSPAK correlated with increased SLC12 cotransporter phosphorylation in WT cells. These preliminary results suggest that tacrolimus promotes WNK body formation and activity via KS-WNK1, and that SPAK localization in HEK cell-reconstituted WNK bodies requires full length WNK1. There is growing evidence that biomolecular condensates concentrate biomolecules to optimize signaling pathways and enhance kinase specificity through spatiotemporal control. Here we propose a novel concept that WNK bodies are biomolecular condensates that are regulated by calcineurin inhibitors. NIH K08DK118211, U54DK137329, ASN Gottschalk Award, P30DK081943. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2.

Inactivating mutations of kidney Na-K-2Cl cotransporter NKCC2 lead to antenatal Bartter syndrome (BS) type 1, a life-threatening salt-losing tubulopathy. We previously reported that this serious inherited renal disease is linked to the endoplasmic reticulum-associated degradation (ERAD) pathway. The purpose of this work is to characterize further the ERAD machinery of NKCC2. Here, we report the identification of ancient ubiquitous protein 1 (AUP1) as a novel interactor of NKCC2 ER-resident form in renal cells. AUP1 is also an interactor of the ER lectin OS9, a key player in the ERAD of NKCC2. Similar to OS9, AUP1 co-expression decreased the amount of total NKCC2 protein by enhancing the ER retention and associated protein degradation of the cotransporter. Blocking the ERAD pathway with the proteasome inhibitor MG132 or the α-mannosidase inhibitor kifunensine fully abolished the AUP1 effect on NKCC2. Importantly, AUP1 knock-down or inhibition by overexpressing its dominant negative form strikingly decreased NKCC2 polyubiquitination and increased the protein level of the cotransporter. Interestingly, AUP1 co-expression produced a more profound impact on NKCC2 folding mutants. Moreover, AUP1 also interacted with the related kidney cotransporter NCC and downregulated its expression, strongly indicating that AUP1 is a common regulator of sodium-dependent chloride cotransporters. In conclusion, our data reveal the presence of an AUP1-mediated pathway enhancing the polyubiquitination and ERAD of NKCC2. The characterization and selective regulation of specific ERAD constituents of NKCC2 and its pathogenic mutants could open new avenues in the therapeutic strategies for type 1 BS treatment.

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.

WNK1 in the kidney.

The aim of this manuscript was to review recent evidence uncovering the roles of the With No lysine (K) kinase 1 (WNK1) in the kidney. Analyses of microdissected mouse nephron segments have revealed the abundance of long-WNK1 and kidney-specific-WNK1 transcripts in different segments. The low levels of L-WNK1 transcripts in the distal convoluted tubule (DCT) stand out and support functional evidence on the lack of L-WNK1 activity in this segment. The recent description of familial hyperkalaemic hypertension (FHHt)-causative mutations affecting the acidic domain of WNK1 supports the notion that KS-WNK1 activates the Na+:Cl- cotransporter NCC. The high sensitivity of KS-WNK1 to KLHL3-targeted degradation and the low levels of L-WNK1 in the DCT, led to propose that this type of FHHt is mainly due to increased KS-WNK1 protein in the DCT. The observation that KS-WNK1 renal protein expression is induced by low K+ diet and recent reassessment of the phenotype of KS-WNK1-/- mice suggested that KS-WNK1 may be necessary to achieve maximal NCC activation under this condition. Evidences on the regulation of other renal transport proteins by WNK1 are also summarized. The diversity of WNK1 transcripts in the kidney has complicated the interpretation of experimental data. Integration of experimental data with the knowledge of isoform abundance in renal cell types is necessary in future studies about WNK1 function in the kidney.

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 sodium/proton exchanger NHA2 regulates blood pressure through a WNK4-NCC dependent pathway in the kidney

NHA2 is a sodium/proton exchanger associated with arterial hypertension in humans, but the role of NHA2 in kidney function and blood pressure homeostasis is currently unknown. Here we show that NHA2 localizes almost exclusively to distal convoluted tubules in the kidney. NHA2 knock-out mice displayed reduced blood pressure, normocalcemic hypocalciuria and an attenuated response to the thiazide diuretic hydrochlorothiazide. Phosphorylation of the thiazide-sensitive sodium/chloride cotransporter NCC and its upstream activating kinase Ste20/SPS1-related proline/alanine rich kinase (SPAK), as well as the abundance of with no lysine kinase 4 (WNK4), were significantly reduced in the kidneys of NHA2 knock-out mice. Invitro experiments recapitulated these findings and revealed increased WNK4 ubiquitylation and enhanced proteasomal WNK4 degradation upon loss of NHA2. The effect of NHA2 on WNK4 stability was dependent from the ubiquitylation pathway protein Kelch-like 3 (KLHL3). More specifically, loss of NHA2 selectively attenuated KLHL3 phosphorylation and blunted protein kinase A- and protein kinase C-mediated decrease of WNK4 degradation. Phenotype analysis of NHA2/NCC double knock-out mice supported the notion that NHA2 affects blood pressure homeostasis by a kidney-specific and NCC-dependent mechanism. Thus, our data show that NHA2 as a critical component of the WNK4-NCC pathway and is a novel regulator of blood pressure homeostasis in the kidney.

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.

Acute and Chronic Effects of Prostaglandin E2 on NKCC2, NCC and ENaC in Ex Vivo Renal Tubules

The major renal prostaglandin, prostaglandin E2 (PGE2), is enzymatically generated from arachidonic acid in a series of steps catalyzed by various enzymes including Cox1/2. PGE2 has modulatory effects on renal Na+ handling, but the molecular targets of these effects in the distal segments of the renal tubule (TAL, DCT, CD) are less well characterized. Here, we assessed acute (30 minutes) and long‐term (24 h) actions of PGE2 (0.1 nM to 10 μM) on various Na+ transport proteins in these segments using tubule suspensions isolated from mouse kidney. In the long‐term, no significant effects could be detected on the abundance of the NaCl cotransporter NCC, the Na‐K‐Cl cotransporter NKCC2 or the epithelial Na+ channel ENaC (αENaC). Altered phosphorylation of NCC (T58) and NKCC2 (T105) or proteolytic cleavage of αENaC was not observed. Similar results were obtained in the presence of Cox1/2 inhibition and no sex differences were observed. Acutely, no effects on NKCC2 or αENaC were observed, but phosphorylation of NCC was decreased. A similar decrease in NCC phosphorylation was detected in NCC‐expressing MDCK cells. Data mining of online kidney RNAseq databases suggested that prostanoid receptor EP4 was expressed in the DCT, and ongoing studies are centered on determining if the actions of PGE2 on NCC are via this receptor.Support or Funding InformationThis project has received funding from The European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie grant agreement no 754513, The Aarhus University Research Foundation and NNF Laureate Research Grant ‐ Novo Nordisk Fonden

Molecular Targets of Natriuretic Action of Prolactin in the Rat Model of Cholestasis of Pregnancy.

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.

Rapid Aldosterone-Mediated Signaling in the DCT Increases Activity of the Thiazide-Sensitive NaCl Cotransporter.

The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. Aldosterone acutely activates NCC to modulate renal NaCl excretion.

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.

Regulation of renal Na transporters in response to dietary K.

Changes in the expression of Na transport proteins were measured in the kidneys of mice with increased dietary K intake for 1 wk. The epithelial Na channel (ENaC) was upregulated, with enhanced expression of full-length and cleaved forms of α-ENaC and cleaved γ-ENaC. At the same time, the amount of the NaCl cotransporter NCC and its phosphorylated form decreased by ~50% and ~80%, respectively. The expression of the phosphorylated form of the Na-K-2Cl cotransporter NKCC2 also decreased, despite an increase in overall protein content. The effect was stronger in males (80%) than in females (40%). This implies that less Na+ is reabsorbed in the thick ascending limb of Henle's loop and distal convoluted tubule along with Cl-, whereas more is reabsorbed in the aldosterone-sensitive distal nephron in exchange for secreted K+. The abundance of the proximal tubule Na/H exchanger NHE3 decreased by ~40%, with similar effects in males and females. Time-course studies indicated that NCC and NHE3 proteins decreased progressively over 7 days on a high-K diet. Expression of mRNA encoding these proteins increased, implying that the decreased protein levels resulted from decreased rates of synthesis or increased rates of degradation. The potential importance of changes in NHE3, NKCC2, and NCC in promoting K+ excretion was assessed with a mathematical model. Simulations indicated that decreased NHE3 produced the largest effect. Regulation of proximal tubule Na+ transport may play a significant role in achieving K homeostasis.

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.

Beta‐Adrenergic Receptor Signaling in Distal Convoluted Tubules

The sympathetic nervous system (SNS) plays a central role in blood pressure regulation. Recent studies have shown that cells of the distal convoluted tubule (DCT) can be stimulated directly via the beta‐adrenergic receptor resulting in activation of the NaCl cotransporter NCC. Whether these effects are mediated by the beta1‐ or beta2‐adrenergic receptor is unclear, and the signaling cascades rapidly activated by beta‐adrenergic receptor stimulation in the DCT are unknown. RT‐PCR identified alpha1B‐, alpha1D‐ and beta2‐adrenergic receptors, but no beta1 receptors, in mpkDCT cells (a model of the DCT). Treatment of mpkDCT cells with the short‐acting beta2‐adrenergic receptor agonist salbutamol (1μM) for 15 min resulted in significantly increased cAMP levels, and phosphorylation of VASP at a PKA‐dependent site. Global changes in signaling networks following 15 min of salbutamol treatment of mpkDCT cells were assessed using stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics. A total of 43773 unique phosphopeptides containing 13599 confidently assigned phosphorylation sites were identified, 9209 of which were suitable for additional statistical analysis. Salbutamol increased phosphorylation at 81 sites; with kinases including PAKB, CLK, p70S6K and PKA predicted to be responsible. 71 sites, including several targets for CDK and GSK3 kinase groups, were decreased in phosphorylation. The predicted kinases fit well with motif analysis, where sites increased in phosphorylation were basophilic (RXXS), whereas the downregulated motif (SXXXS) is predicted to be a target for GSK3. Pathway analysis predicted that CREB and AKT are also important pathway nodes altered by salbutamol. Phosphorylation of the NCC‐modulating kinase OSR1 was increased at S339, a site previously shown to be regulated by PI3K‐mediated signaling. In summary, beta2‐adrenergic receptor signaling in mpkDCT cells involves PI3K and cAMP‐mediated pathways. Incorporation of our data into known signaling networks may provide novel clues linking the sympathetic nervous system, DCT regulation and NCC activity.Support or Funding InformationFunding is provided by the Novo Nordisk Foundation, the Lundbeck Foundation, Marie Skłodowska‐Curie Individual Fellowship and the Danish Medical Research Council.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.