Significance Statement The calcium-sensing receptor (CaSR) activates salt reabsorption through the sodium-chloride cotransporter (NCC) via the WNK4-SPAK pathway. Glucose and other sugars are positive allosteric modulators of the CaSR. This would be irrelevant for most cells that come into contact with glucose, except for the nephron segments beyond the proximal tubule. Using in vitro and in vivo models, this work shows that the arrival of glucose or fructose to the distal nephron modulates the CaSR due to a positive allosteric type II effect associated with activation of the WNK4-SPAK-NCC pathway. This study proposes a mechanism by which glucose or fructose delivered to the distal nephron contributes to renal salt retention by their allosteric effect on the CaSR. Background The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. Methods We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. Results HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. Conclusions Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule, and the inhibition of its function with thiazides is widely used for the treatment of arterial hypertension. In mammals and teleosts, NCC is present as one ortholog that is mainly expressed in the kidney. One exception, however, is the eel, which has two genes encoding NCC. The eNCCα is located in the kidney and eNCCβ, which is present in the apical membrane of the rectum. Interestingly, the European eNCCβ functions as a Na+-Cl- cotransporter that is nevertheless resistant to thiazides and is not activated by low-chloride hypotonic stress. However, in the Japanese eel rectal sac, a thiazide-sensitive NaCl transport mechanism has been described. The protein sequences between eNCCβ and jNCCβ are 98% identical. Here, by site-directed mutagenesis, we transformed eNCCβ into jNCCβ. Our data showed that jNCCβ, similar to eNCCβ, is resistant to thiazides. In addition, both NCCβ proteins have high transport capacity with respect to their renal NCC orthologs and, in contrast to known NCCs, exhibit electrogenic properties that are reduced when residue I172 is substituted by A, G, or M. This is considered a key residue for the chloride ion-binding sites of NKCC and KCC. We conclude that NCCβ proteins are not sensitive to thiazides and have electrogenic properties dependent on Cl-, and site I172 is important for the function of NCCβ.

As a result of the pandemic, cases requiring endotracheal intubation have increased to support breathing through mechanical ventilation; this procedure is necessary when a patient cannot breathe on their own due to respiratory failure. For it, the laryngoscope is used. Even when using a laryngoscope, it is possible to injure the patient due to poor visibility at intubation. As a solution, some video-laryngoscopes present many advantages, such as reducing injuries, lowering the number of attempts to secure the airway and the image’s quality, and easy recognition of laryngeal structures. However, this type of device is not accessible to many health institutions due to the price. Therefore, this study proposes the design of a video-laryngoscope in the Computer-Aided-Design (CAD) software and constructed by additive manufacturing. For this design, the identification of the needs of the medical personnel in the anesthesiology and emergency medicine departments from the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ) was taken into account. The design specifications provided by the users were used to create a proposal solution at the conceptual design level. The material and camera for the prototype instrumentation are presented with the model’s geometric parameters. Subsequently, virtual modeling of the device was carried out, and rapid prototypes were built to validate the performance of the design proposal. The final device was constructed by Selective Laser Sintering (SLS) using Nylon 12 as a construction material. Finally, the device was evaluated using airway simulators with the support of medical specialists to determine its functionality.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2 -terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels. NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K + and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.

BackgroundEdema formation in patients with chronic liver, heart or kidney disease is associated with salt and water retention by the kidneys. The molecular pathways in the nephron that lead to this event in humans remain elusive. Furthermore, the pattern of salt retention pathways enhanced can be different depending on the type of edematous state. The use of urinary exosomes (UE) is a powerful non‐invasive tool to study the pathophysiological state of the kidney in many diseases and its potential as a diagnostic tool for assessment of human samples is rising. The objective of our study was to assess the expression of different renal transporters in urinary exosomes of patients with chronic failure of the heart, the liver or the kidney, with and without edema.MethodsWe conducted a prospective and observational study where we obtained clinical and biochemical data, as well as urinary samples from adult patients with liver cirrhosis (Child B–C) (N=9 with, N=10 no‐edema), chronic kidney disease (CKD; KDIGO G3–G4) (N=9 with, N=18 no‐edema), chronic heart failure (NYHA II–III) (N=5 with, N=5 no‐edema), and healthy volunteers (N=4) as the control group. We isolated UE from 8 ml of urine with a two‐step ultracentrifugation process and analyzed with western blot the expression of SGLT2, NHE3, NKCC2, NCC and αENaC. The amount of UE used per patient was adjusted to urinary creatinine.ResultsWe found no significant differences in laboratory findings of cirrhotic patients with or without edema, except for lower serum Na+ in the edema group (133.9±4.9 vs. 140.1±2.1 mEq/l p<0.05). In CKD patients, however, we found differences in the BMI (31±6.9 vs. 25±3 p<0.01), serum K+ (5.2±0.39 vs. 4.6±0.55 mEq/l p<0.01) and Ca2+ (8.6±0.47 vs. 9.3±0.75 mg/dl p<0.05) in the edema vs. no‐edema group, respectively. In heart failure patients we observed differences in serum Na+ (133.8±4.9 vs. 138.6±4.4 mEq/l, p<0.05). Analysis of UE showed a significant increase of SGLT2 and NHE3 expression in cirrhotic patients with edema and in heart failure patients with and without edema, when compared to control group. Patients with heart failure also exhibited increased expression of NKCC2 and αENaC. Interestingly, in both cirrhotic and heart failure patients NCC expression was down‐regulated. In CKD patients, we observed increased expression of SGLT2, NHE3 and αENaC.DiscussionCompared to healthy volunteers, all pathological conditions have upregulation of certain renal transporters. We observed different expression patterns of UE for each pathologic state. Decreased expression of NCC seems to be a compensatory mechanism. These results suggest that the analysis of renal transporters using UE can give insights into the molecular mechanisms of salt retention in patients with edematous states and may contribute to the design of early therapeutic strategies.Support or Funding InformationConacyt‐Fosiss grant No. 261562 to GGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Hypercalciuria can result from activation of the basolateral calcium-sensing receptor (CaSR), which in the thick ascending limb of Henle's loop controls Ca2+ excretion and NaCl reabsorption in response to extracellular Ca2+ However, the function of CaSR in the regulation of NaCl reabsorption in the distal convoluted tubule (DCT) is unknown. We hypothesized that CaSR in this location is involved in activating the thiazide-sensitive NaCl cotransporter (NCC) to prevent NaCl loss.Methods We used a combination of in vitro and in vivo models to examine the effects of CaSR on NCC activity. Because the KLHL3-WNK4-SPAK pathway is involved in regulating NaCl reabsorption in the DCT, we assessed the involvement of this pathway as well.Results Thiazide-sensitive 22Na+ uptake assays in Xenopus laevis oocytes revealed that NCC activity increased in a WNK4-dependent manner upon activation of CaSR with Gd3+ In HEK293 cells, treatment with the calcimimetic R-568 stimulated SPAK phosphorylation only in the presence of WNK4. The WNK4 inhibitor WNK463 also prevented this effect. Furthermore, CaSR activation in HEK293 cells led to phosphorylation of KLHL3 and WNK4 and increased WNK4 abundance and activity. Finally, acute oral administration of R-568 in mice led to the phosphorylation of NCC.Conclusions Activation of CaSR can increase NCC activity via the WNK4-SPAK pathway. It is possible that activation of CaSR by Ca2+ in the apical membrane of the DCT increases NaCl reabsorption by NCC, with the consequent, well known decrease of Ca2+ reabsorption, further promoting hypercalciuria.

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Objective: The hypertensive effect of angiotensin II (AngII), a peptide hormone, is dependent on its intrarenal actions and the activation of the renal Na-Cl cotransporter (NCC), by AngII requires integrity of the with no lysine kinase/STE20-proline alanine-rich kinase (WNK/SPAK) signaling pathway. Here, we analyzed if the integrity of the WNK/SPAK pathway is required for AngII infusion to induce arterial hypertension. Methods: We tested the effect of AngII or aldosterone administration on the blood pressure and on pNCC/NCC ratio in SPAK T243A/243A knock-in mice in which the kinase and thus NCC cannot be activated by WNK kinases. AngII or aldosterone was infused at 1440 or 700mg/kg per day, respectively, for 14 days using osmotic minipumps. The aldosterone-treated mice were exposed to NaCl drinking water (1%) during the hormone administration. The arterial blood pressure was assessed using radiotelemetry. Results: We observed that in the SPAK knock-in mice, the AngII-induced hypertensive effect was significantly reduced and associated with an absence of AngII-induced NCC phosphorylation. In contrast, the hypertensive effect of aldosterone was enhanced and was related with an increased response to amiloride, but not to thiazide-type diuretics, without a significant increase in NCC phosphorylation. Conclusion: Our data suggest that AngII-induced hypertension requires, at least partly, NCC activation via the WNK/SPAK signaling pathway, whereas aldosterone-induced hypertension depends on epithelial sodium channel activation in a WNK/SPAK-independent manner. SPAK knock-in mice emerge as a useful model to distinguish between the effects of AngII and aldosterone on distal nephrons.

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

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.

The renal thiazide-sensitive NaCl cotransporter, NCC, is the major pathway for salt reabsorption in the distal convoluted tubule. The activity of this cotransporter is critical for regulation of several physiological variables such as blood pressure, serum potassium, acid base metabolism, and urinary calcium excretion. Therefore, it is not surprising that numerous hormone-signaling pathways regulate NCC activity to maintain homeostasis. In this review, we will provide an overview of the most recent evidence on NCC modulation by aldosterone, angiotensin II, vasopressin, glucocorticoids, insulin, norepinephrine, estradiol, progesterone, prolactin, and parathyroid hormone.

Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na+-Cl- cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.

Tacrolimus‐induced hypertension in mice is associated with increased activity of the renal NaCl cotransporter (NCC). It is not known if this occurs in humans. Here, we performed a prospective, longitudinal and observational study to assess the prevalence of hypertension in patients undergoing renal transplantation and the expression/phosphorylation of NCC in urinary exosomes. From January 2013 all adult patients receiving a kidney allograft were enrolled. All patients received tacrolimus. At six and 12 months after surgery we assessed general clinical and laboratory variables, tacrolimus blood levels, and arterial blood pressure, which at six months was determined by ambulatory blood pressure monitoring (ABPM). At six and twelve months urinary exosomes were extracted to perform Western blot analysis using total and phospho‐NCC antibodies (T45‐50‐55‐NCC). At the moment 43 patients (69% male) have completed six months of follow up and ABPM. Mean age 36.7 ± 13.5 years. Of 17 women three had hypertension (17%), while of 26 males, 15 had hypertension (57%). Hypertensive men were older than normotensive (44.2 ± 14.2 vs. 30.7 ± 9.7, p<0.001), but body mass index, allograft rejection rate, serum tacrolimus and creatinine levels were similar. Western blot analysis has been performed in urinary exosomes from 9 hypertensive and 5 normotensive men. Values in normotensive men were taken as 1 and in hypertenvise normalized accordingly. Values in hypertensive vs. normotensive were: for NCC expression: 2.94 ± 0.5 vs. 1.0 ± 0.2 and pNCC 1.8 ± 0.4 vs. 1.0 ± 0.3, respectively (p<0.05). Our data suggest that NCC expression/phosphorylation is increased in hypertensive postranplant patients treated with tacrolimus.

Evidence in rodents suggests that tacrolimus-induced posttransplant hypertension is due to upregulation of the thiazide-sensitive Na+-Cl- cotransporter NCC. Here, we analyzed whether a similar mechanism is involved in posttransplant hypertension in humans. From January 2013 to June 2014, all adult kidney transplant recipients receiving a kidney allograft were enrolled in a prospective cohort study. All patients received tacrolimus as part of the immunosuppressive therapy. Six months after surgery, we assessed general clinical and laboratory variables, tacrolimus trough blood levels, and ambulatory 24-h blood pressure monitoring. Urinary exosomes were extracted to perform Western blot analysis using total and phospho-NCC antibodies. A total of 52 patients, including 17 women and 35 men, were followed. At 6 mo after transplantation, of the 35 men, 17 developed hypertension and 18 remained normotensive, while high blood pressure was observed in only 3 of 17 women. The hypertensive patients were significantly older than the normotensive group; however, there were no significant differences in body weight, history of acute rejection, renal function, and tacrolimus trough levels. In urinary exosomes, hypertensive patients showed higher NCC expression (1.7±0.19) than normotensive (1±0.13) (P=0.0096). Also, NCC phosphorylation levels were significantly higher in the hypertensive patients (1.57±0.16 vs. 1±0.07; P=0.0049). Our data show that there is a positive correlation between NCC expression/phosphorylation in urinary exosomes and the development of hypertension in posttransplant male patients treated with tacrolimus. Our results are consistent with the hypothesis that NCC activation plays a major role in tacrolimus-induced hypertension.

It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.

The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine–rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice (WNK1+/FHHt) with WNK4−/− mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1+/FHHt mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine–rich kinase–dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine–rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice ( WNK1 +/FHHt ) with WNK4 −/− mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1 +/FHHt mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine–rich kinase–dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

Pregnancy, lactation and the female sexual cycle are physiological conditions in which volume retention is required. It has been shown that the renal thiazide‐sensitive NaCl cotransporter (NCC) expression is higher in female than in male rats, but little is known about the mechanism. In this study we observed that, in contrast to male rats, female rats exhibit a higher response to a single ip injection of a thiazide type diuretic, but not to furosemide or amiloride. In the kidney, female rat and mice exhibit higher expression and T‐58 phosphorylation of NCC than males, as well as higher expression and S‐383 phosphorylation of the kinase SPAK. The difference disappeared after ovariectomy. No effect of orquiectomy was observed. The administration of β‐estradiol or progesterone in ovariectomized rats restored the increased phosphorylation of NCC. A role for prolactin in NCC regulation, however, cannot be ruled out because estrogens enhance prolactin secretion. In addition, prolactin is a known osmoregulator in teleost fish. We thus assessed the expression and phosphorylation of NCC in mice lacking the prolactin receptor (PRLR‐/‐). The difference between female and male persisted in PRLR‐/‐ mice, but this could be due to ovarian hormones. However, in male PRLR‐/‐ mice phosphorylation of NCC was dramatically reduced, when compared to PRLR+/+ littermates, suggesting that by lacking estrogen and progesterone, male PRLR‐/‐ mice are more suitable model to document the NCC regulation by prolactin. Supporting this hypothesis, ex vivo male rat kidneys perfusion with prolactin demonstrated a dramatic increase in NCC phosphorylation when compared with vehicle‐perfused controls. We thus conclude that estrogen, progesterone, and prolactin are positive activators of NCC, which in turn play a role in the volume retention observed in female physiological conditions such as the female sexual cycle, pregnancy and lactation.

Modulation of Na+-Cl- cotransporter (NCC) activity is essential to adjust K+ excretion in the face of changes in dietary K+ intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine- rich kinase (SPAK) and with-no-lysine kinase (WNK) 4 in the modulation of NCC by K+ diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K+ -citrate diets for 4 days. The low-K+ diet decreased and high-K+ diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr(44)/Thr(48)/Thr(53)) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho- SPAK/OSR1, Ser(383)/Ser(325)). The effect of the low-K+ diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K+ diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K+ diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K+ -citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K+ increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K+ diet.